AU2012234682A1

AU2012234682A1 - Inhibitors of 17beta-HSD1, 17beta-HSD3 and 17beta-HSD10

Info

Publication number: AU2012234682A1
Application number: AU2012234682A
Authority: AU
Inventors: Rene Maltais; Donald Poirier; Jenny Roy
Original assignee: Universite Laval
Current assignee: Universite Laval
Priority date: 2011-03-25
Filing date: 2012-03-26
Publication date: 2013-10-10
Also published as: CA2830984C; EP2688901A4; JP2018062523A; JP2014508784A; EP2688901B1; CA2830984A1; JP6727184B2; WO2012129673A1; EP2688901A1; US11072632B2; US20140088053A1

Abstract

The present application discloses 17β hydroxy steroid dehydrogenase (17β HSD) type 1, 3, 10 inhibitors and use thereof (alone and in combination) in the treatment of cancer and other afflictions. 17β HSDl inhibitors include estradiol derivatives with a nieta-carbamoylbenzyl substituent at C 16. 17β HSD3/HSD10 inhibitors include androsterone derivatives substituted at the C3 position with a sulfonamide piperazine. Also disclosed are compounds that are inhibitors of both 17β HSDl and 17β HSD3 that have a spiro-morpholine substituent at C20.

Description

WO 2012/129673 PCT/CA2012/000316 TITLE INHIBITORS OF 17p-HSD1, 17p-HSD3 and 17p-HSD1O. FIELD [0001] The present description broadly relates to inhibitors of at least one of 17p-HSDI, 17p-HSD3 and 17p-HSD10. BACKGROUND [00021 17p-Hydroxysteroid dehydrogenase type 1 (17p-HSDI) transforms estrone (El) into estradiol (E2), the most potent natural ligand for the estrogen receptor (ER). This enzyme also catalyzes the reduction of dehydroepiandrosterone (DHEA) into 5-androstene-3p,17-diol (A 5 -diol), a weaker estrogen but especially important after menopausis.

1

,

2 Inhibitors of 17p-HSDI are thus interesting therapeutic agents for the control of estrogen-dependent diseases such as breast cancers and endometriosis.

3

,

4 During the last thirty years, intense efforts were deployed with the goal of designing potent inhibitors of this key steroidogenic enzyme5- but, it is only recently that lead candidates have been reported with very good inhibitory activities.

9

-

10 The presence of a residual estrogenic activity associated with steroidal inhibitors, which are often built around an estrane scaffold," represented a major drawback in their development. [00031 16p-(m-carbamoylbenzyl)estradiol (I) has been reported as a potent inhibitor of 17p-HSD12. Despite of its good inhibitory potency, I was found to stimulate in vitro both the MCF-7 and T-47D estrogen-sensitive breast cancer cell lines, thus greatly reducing its therapeutic potential. 10004] In order to eliminate its undesirable residual estrogenic activity, the impact on both 17p-HSDI inhibition and estrogenicity by positioning small chains differently functionalized in substitution of the phenol group at position C3 has been WO 2012/129673 PCT/CA2012/000316 -2 previously explored. The choice of replacing the phenolic group of I by a 3-alkyl chain was guided from X-ray analysis of the crystallized complex of inhibitor I with 17p-HSD1, which shows key interactions for the inhibitory activity (Scheme 1).1 In fact, three major interactions were identified in the binary complex of 17p-HSDI and inhibitor I): the 17p-OH forms a hydrogen bond with the Ser142; the CONH 2 group forms a hydrogen bond with Phe192; and the phenyl ring at C16 forms a t-7E interaction with Tyr 155. However, contrary to El, the natural substrate of the enzyme, the 3-OH of I does not form hydrogen bonding with either Glu282 or His221. Ser' 4 2 Tyr1 55 OH Phe 92

NH

2 OH H H H NH 2 ' R H H HO R = CH 2 OH (11) CH 2

CH

2 OH (IV)

CH

2 Br (Ill) CH 2

CH

2 Br (V) Scheme 1: Key Interactions for the Inhibitory Activity of 17p-HSD1 [00051 Prostate cancer is the most common cancer among United States men with an estimated 217730 new diagnosed cases in 2010 and with 32050 associated deaths.

14 Endocrine therapy has been recognized as one of the most efficient treatments for prostate cancer with a positive response in cancer regression in nearly of 80% of the cases following a first treatment.

15 Different endocrine treatments are now available to block either the testicular source of androgens (medical or surgical castration) or to block the effect of androgen testosterone (T) and dihydrotestosterone (DHT) on the androgen receptor (antiandrogens).16" 7 Although it improves survival, these androgen deprivation treatments are associated with important side effects and are not curative in cases of advanced prostate cancer.1-2 Thus, the development of new therapeutic options are strongly needed to improve survival as well as the life quality of treated patients.

WO 2012/129673 PCT/CA2012/000316 -3 [0006] 17p-hydroxysteroid dehydrogenase type 3 (17p-HSD3), also named testicular 17p-hydroxysteroid dehydrogenase, is a steroidogenic enzyme that catalyzes the reduction of non-androgenic 4-androstene-3,17-one (A 4-dione)2 to potent androgen T using NADPH as cofactor. 9,23-2 This enzyme is found primarily in the Leydig cells in the microtubule part of the testis and contributes to the production of approximately 60% of total active androgens in men.26 The other 40% of active androgens would be directly synthesized in the prostate from the inactive adrenal precursors dehydroepiandrosterone (DHEA) and A 4 -dione by the action of 3p hydroxysteroid dehydrogenases, 5a-reductases and other 17p-HSDs such as type 5 or type 15.27,28 However, although the 17p-HSD3 expression level is very low in normal prostate tissue, it has been reported that 17p-HSD3 is suspected to play an important role in the conversion of adrenal steroids into potential androgens in prostate cancer tissue.

29

,

30 In fact, the expression level of 17p-HSD3 mRNA in prostatic tissue with malignancy is significantly higher (31 times) than those in prostatic tissue without malignancy. Furthermore, it was recently shown that 17p-HSD3 is overexpressed (8 times) in the LuCaP 23 and LuCAP 35 cell lines isolated from metastatic tissue obtained from patients resistant to castration therapy. 3-34 Importantly, despite a castrated level of T in the bloodstream, the level of T within the metastatic tumors was found to be sufficiently high to stimulate the proliferation of cancer cells. 100071 17p-hydroxysteroid dehydrogenase type 10 (17p-HSD1O) is a mitochondrial enzyme involved in estrogen inactivation, androgen activation, p oxidation of fatty acids and isomerisation of bile acids. Since this enzyme uses estradiol (E2) as a substrate, there is evidence that the enzyme contributes contribute to Alzheimer's disease pathogenesis by reducing neuroprotective estrogen levels. Moreover, this enzyme plays a significant role in a non-classical androgen synthesis pathway and its expression is up-regulated in certain prostate cancer cells, thus conferring an advantage upon these cells for surviving androgen ablation therapy.

6

'

35 37 Consequently, the inhibition of 17p-HSDI0 provides a new approach to the treatment of these diseases.

WO 2012/129673 PCT/CA2012/000316 -4 [00081 The present specification refers to a number of documents, the content of which is herein incorporated in their entirety. SUMMARY [00091 The present disclosure relates to inhibitors of 17p-HSDI, 170 HSD3 and 17p-HSDI0. In an embodiment, the present disclosure relates to inhibitors of at least one of 17-HSD1, 17p-HSD3 and 17p-HSDIO. In an embodiment, the present disclosure relates to selective inhibitors of 17p-HSDI, 17P-HSD3 or 17p HSDI0. In an embodiment of the present disclosure, the inhibitors exhibit a non estrogenic (17p-HSDI) or non-androgenic profile (17p-HSD3). BRIEF DESCRIPTION OF THE DRAWINGS [00101 In the appended figures: [0011] FIG. 1 is an illustration of the effect of inhibitors I-V on the growth of estrogen-starved estrogen sensitive MCF-7 cells after 7 days of treatment at different concentrations. The proliferation of control cells is fixed at 100%. Results are expressed as means ± SEM of triplicate. * P < 0.05 versus control. ** P < 0.01 versus control. [0012] FIG. 2 is an illustration of a competitive binding assay of Compound V and estradiol in a human estrogen receptor assay. [0013] FIG 3 shows the inhibitory potency of Compound V and I in T 47D intact cells. Results are the means (± SEM) of a triplicate. [0014] FIG. 4. A shows the cell growth of T-47D cells induced by a physiologic concentration of El (0.1 nM) in the presence or absence of inhibitors Compounds I and V at various concentrations. Results are expressed as means (+ WO 2012/129673 PCT/CA2012/000316 -5 SEM) of triplicate. B: Effect of inhibitor Compound V and the pure antiestrogen EM 139 on the inhibition of E2 (0.1 nM)- induced proliferation (antiestrogenic activity) of estrogen-sensitive (ER*) human breast cancer T-47D cells. Results are expressed as means (± SEM) of triplicate. [0015] FIG5. A shows the effects of inhibitors Compounds I and V on the growth of estrogen-starved T-47D (ERT) human breast cancer cells after 7 days of treatment.. Results are expressed as means (+ SEM) of triplicate (*P < 0.05 and **P < 0.01). B: Effects of increasing concentrations of Compounds I and V in displacing

[

3 H]-E2 binding to the human ERa. 10016] FIG. 6. shows the effect of inhibitors Compounds I and V on uterine (A) and vagina (B) weight of ovariectomized (OVX) mice treated for 7 days (*P < 0.05 and **P < 0.01, experimental versus OVX control animals (CTR)). [00171 FIG. 7 shows the plasma concentration of Compounds I and V as a function of time following subcutaneous (s.c.) injection of 2.3 mg/kg in Sprague Dawley rats. 10018] FIG. 8 shows the effect of Compound V on the growth of El (s.c.)-stimulated T-47D tumors (xenograft) in ovariectomized (OVX) nude mice. (*P < 0.05 and **P < 0.01, El-Compound V and OVX control animals (CTR) versus El). 10019] FIG. 9. shows the effect of inhibitor of Compound V and estrone (El) on the body (A), uterine (B) and vaginal (C) weight of ovariectomized nude mice after 32 days of treatment. CTR: control. [0020] FIG. 10 illustrates a schematic view of the contribution of 17p HSD3 in androgen testostosterone (T) biosynthesis from the non-androgenic 4 androstene-3,17-dione

(A

4 -dione) in different sources including the testis (endocrine), WO 2012/129673 PCT/CA2012/000316 -6 the adrenals (endocrine), and intratumoral tissues (intracrine). LH: Luteinizing hormone, 5a-R: 5a-reductase, AR: androgen receptor, DHT: dihydrotestosterone, 17p-HSD5 and 17-HSDI 5: types 5 and 15 of 17p-hydroxysteroid dehydrogenase. 100211 FIG. 11A is an illustration of the relative importance of different chemical groups (i.e. alkyl, alkylamide and carbamate derivatives) attached to position 3P of the androsterone steroid scaffold and their effect on 17p-HSD3 inhibition as established by SAR studies. FIG. IB is an illustration of representative inhibitors of 17p-HSD3. [0022] FIG. 12 is an illustration of the inhibition of the transformation of [14C]-4-androstene-3,17-dione ([1 4

C]-A

4 -dione) (50 nM) into [14C]-testosterone ([14C] T) by 17j-HSD3 overexpressed in intact HEK-293 cells. [0023] FIG. 13 is an illustration of the proliferative effect (androgenic activity) on androgen sensitive Shionogi cells induced by potent androgen dihydrotestosterone (DHT) and the 170-HSD3 inhibitors CS-213, 5, 7a, 15b and 17a. The data are expressed as means ± SEM of one experiment in triplicate. Significantly different from control (CTL): * (p < 0.01). 100241 FIG. 14A is an illustration of the proliferative (estrogenic) effect of compounds (1-4) on MCF-7 (ER*) cells growth at different concentrations (< I tM). (B) Cytotoxic effect of compounds (1-4) on MCF-7 (ER) cells at high concentrations (> I pM). The cell proliferation without product was fixed as 100%. The relative proliferative potency (RPP) or relative cytotoxic potency (RCP) were calculated as IC 50 (17p-E2)/lC50 (compound 1, 2,3 or 4) X 100 100251 FIG. 15A is an illustration of the proliferative (estrogenic) effect of compounds 1-4 on T-47D (ER*) cells at different concentrations (< I prM). (B) Cytotoxic effect of compounds 1-4 on T-47D (ER*) cells at high concentrations (> I WO 2012/129673 PCT/CA2012/000316 -7 [M). The cell proliferation without product was fixed as 100%. The relative proliferative potency (RPP) or relative cytotoxic potency (RCP) was calculated as

IC

50 (17p-E2)/IC50 (compound 1, 2, 3 or 4) X 100. [0026] FIG. 16 is an illustration of the effect of compounds 1-4 on BT-20 (ER~) cells growth at different concentrations. [0027] FIG. 17 is an illustration of the effect of increasing concentrations of 17p-E2 (1), 17a-E2 (2), 18-epi-173-E2 (3) and 18-epi-17a-E2 (4) in displacing

[

3 H]-17p-E2 binding to the rat uterine estrogen receptor alpha (ERa). RBA: relative binding affinity. [0028] FIG. 18 is an illustration of Uterine (A) and Vagina (B) weight of mice treated 7 days with 1, 10 and 100 pg/kg (s.c. BID) of compound 1, 2, 3 or 4. * P< 0.05 vs control, **P< 0.01 versus control. [0029] FIG. 19 represents an illustration of the Overlapping of the four E2 isomers (compounds 1-4) showing the positioning of the key 17-OH group. 17 E2 (1) in red, 17a-E2 (2) in blue, 18-epi-17p-E2 (3) in green and 18-epi-17a-E2 (4) in pink. The structure energy minimization as well as overlaying were performed using Chem3D (Pro Version 5.0) software. The energy minimization were obtained with the MM2 minization method (RMS gradient = 0.100). [0030] FIG. 20 illustrates the reactions catalyzed by 17p-HSD1O and cofactors NAD+ (oxidation) or NADH (reduction). 10031] FIG. 21 illustrates the inhibition of transfected 17p-HSDIO in intact HEK-293 cells by compound 21 and E2. Compounds were tested at various concentrations to determine IC 50 values. When the error bars are not shown, it is because they are smaller than the symbol.

WO 2012/129673 PCT/CA2012/000316 -8 DETAILED DESCRIPTION 100321 In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure pertains. [00331 The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more. [0034] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. [00351 The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value. 10036] The terms "acyl" or "alkanoyl," as used interchangeably herein, represent an alkyl group, as defined herein, or hydrogen attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl, acetyl, propionyl, butanoyl and the like. Exemplary unsubstituted acyl groups comprise from 2 to 10 carbons.

WO 2012/129673 PCT/CA2012/000316 -9 100371 The term "alkyl" or "alk" as used herein, represents a monovalent group derived from a straight or branched chain saturated hydrocarbon comprising, unless otherwise specified, from 1 to 15 carbon atoms and is exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl and the like and may be optionally substituted with one, two, three or, in the case of alkyl groups comprising two carbons or more, four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) alkynyl of two to six carbon atoms; (5) amino; (6) aryl; (7) arylalkoxy, where the alkylene group comprises one to six carbon atoms; (8) azido; (9) cycloalkyl of three to eight carbon atoms; (10) halo; (11) heterocyclyl; (12) (heterocycle)oxy; (13) (heterocycle)oyl; (14) hydroxyl; (15) hydroxyalkyl of one to six carbon atoms; (16) N-protected amino; (17) nitro; (18) oxo or thiooxo; (19) perfluoroalkyl of 1 to 4 carbon atoms; (20) perfluoroalkoxyl of I to 4 carbon atoms; (21) spiroalkyl of three to eight carbon atoms; (22) thioalkoxy of one to six carbon atoms; (23) thiol; (24) OC(O)RA, where RA is selected from the group consisting of (a) substituted or unsubstituted C 1

.

6 alkyl, (b) substituted or unsubstituted C 6 or Cio aryl, (c) substituted or unsubstituted C7-16 arylalkyl, where the alkylene group comprises one to six carbon atoms, (d) substituted or unsubstituted C 1

.

9 heterocyclyl, and (e) substituted or unsubstituted C 2 15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (25) C(O)RB, where RB is selected from the group consisting of (a) hydrogen, (b) substituted or unsubstituted CI- 6 alkyl, (c) substituted or unsubstituted C 6 or CIO aryl, (d) substituted or unsubstituted C 7

_

16 arylalkyl, where the alkylene group comprises one to six carbon atoms, (e) substituted or unsubstituted C 1

.

9 heterocyclyl, and (f) substituted or unsubstituted C 2

-

15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (26) CO 2 RB, where RB is selected from the group consisting of (a) hydrogen, (b) substituted or unsubstituted CI.6 alkyl, (c) substituted or unsubstituted C 6 or CIO aryl, (d) substituted or unsubstituted C 7

-

16 arylalkyl, where the alkylene group comprises one to six carbon atoms, (e) substituted or unsubstituted

C

1

.

9 heterocyclyl, and (f) substituted or unsubstituted C 2 15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (27) C(O)NRCRD, where each WO 2012/129673 PCT/CA2012/000316 -10 of RC and RD is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, where the alkylene group comprises one to six carbon EE atoms; (28) S(O)R , where RE is selected from the group consisting of (a) alkyl, (b) aryl, (c) arylalkyl, where the alkylene group comprises one to six carbon atoms, and EE (d) hydroxyl; (29) S(O) 2 R , where RE is selected from the group consisting of (a) alkyl, (b) aryl, (c) arylalkyl, where the alkylene group comprises one to six carbon atoms, and (d) hydroxyl; (30) S(O) 2 NRFRG, where each of RF and RG is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; and (31) NRH R, where each of RH and R' is independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, where the alkylene group comprises one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms, (i) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms, and the alkylene group comprises one to ten carbon atoms, (j) alkanoyl of one to six carbon atoms, (k) aryloyl of 6 to 10 carbon atoms, (1) alkylsulfonyl of one to six carbon atoms, and (m) arylsulfonyl of 6 to 10 carbons atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group. 100381 The terms "alkoxy" or "alkyloxy," as used interchangeably herein, represent an alkyl group attached to the parent molecular group through an oxygen atom. 100391 The term "alkylsulfinyl" as used herein, represents an alkyl group attached to the parent molecular group through an S(O) group. [00401 The term "alkylsulfonyl," as used herein, represents an alkyl group attached to the parent molecular group through a S(O) 2 group.

WO 2012/129673 PCT/CA2012/000316 - II 100411 The term "alkylthio" as used herein, represents an alkyl group attached to the parent molecular group through a sulfur atom. [00421 The term "alkylene" as used herein, represents a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene and the like. [00431 The term "alkenyl," as used herein, represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 15 carbons, such as, for example, 2 to 6 carbon atoms or 2 to 4 carbon atoms, containing one or more carbon-carbon double bonds and is exemplified by ethenyl, I -propenyl, 2-propenyl, 2 methyl- 1 -propenyl, I-butenyl, 2-butenyl and the like and may be optionally substituted with one, two, three or four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) alkynyl of two to six carbon atoms; (5) amino; (6) aryl; (7) arylalkoxy, where the alkylene group comprises one to six carbon atoms; (8) azido; (9) cycloalkyl of three to eight carbon atoms; (10) halo; (11) heterocyclyl; (12) (heterocycle)oxy; (13) (heterocycle)oyl; (14) hydroxyl; (15) hydroxyalkyl of one to six carbon atoms; (16) N-protected amino; (17) nitro; (18) oxo or thiooxo; (19) perfluoroalkyl of 1 to 4 carbon atoms; (20) perfluoroalkoxyl of I to 4 carbon atoms; (21) spiroalkyl of three to eight carbon atoms; (22) thioalkoxy of one to six carbon atoms; (23) thiol; (24) OC(O)R , where RA is selected from the group consisting of (a) substituted or unsubstituted CI- 6 alkyl, (b) substituted or unsubstituted C 6 or Cio aryl, (c) substituted or unsubstituted C 7

-

1 6 arylalkyl, where the alkylene group comprises one to six carbon atoms, (d) substituted or unsubstituted C 1

.

9 heterocyclyl, and (e) substituted or unsubstituted C 2

-

15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (25) C(O)RB, where RB is selected from the group consisting of (a) hydrogen, (b) substituted or unsubstituted C1.

6 alkyl, (c) substituted or unsubstituted C 6 or CIO aryl, (d) substituted or unsubstituted C 7

.

16 arylalkyl, where the alkylene group comprises WO 2012/129673 PCT/CA2012/000316 - 12 one to six carbon atoms, (e) substituted or unsubstituted CI.

9 heterocyclyl, and (f) substituted or unsubstituted C 2

-

15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (26) CO 2 RB, where RB is selected from the group consisting of (a) hydrogen, (b) substituted or unsubstituted CI- 6 alkyl, (c) substituted or unsubstituted C 6 or C 1 o aryl, (d) substituted or unsubstituted C 7

.

C

1

.

9 heterocyclyl, and (f) substituted or unsubstituted C 2 -is heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (27) C(O)NRCRD, where each of RC and RD is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; (28) S(O)RE, where RE is selected from the group consisting of (a) alkyl, (b) aryl, (c) arylalkyl, where the alkylene group comprises one to six carbon atoms, and (d) hydroxyl; (29) S(O) 2 RE, where RE is selected from the group consisting of (a) alkyl, (b) aryl, (c) arylalkyl, where the alkylene group comprises one to six carbon atoms, and (d) hydroxyl; (30) S(O) 2 NRFRG, where each of RF and RG is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; and (31) NRH R, where each of RH and R' is independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, where the alkylene group comprises one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms; (i) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms, and the alkylene group comprises one to ten carbon atoms, (j) alkanoyl of one to six carbon atoms, (k) aryloyl of 6 to 10 carbon atoms, (1) alkylsulfonyl of one to six carbon atoms, and (m) arylsulfonyl of 6 to 10 carbons atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group. [0044] The term "alkynyl" as used herein, represents monovalent straight or branched chain groups of from two to six carbon atoms comprising a carbon carbon triple bond and is exemplified by ethynyl, 1-propynyl, and the like and may be WO 2012/129673 PCT/CA2012/000316 - 13 optionally substituted with one, two, three or four substituents independently selected from the group consisting of: (1) alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to six carbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) alkynyl of two to six carbon atoms; (5) amino; (6) aryl; (7) arylalkoxy, where the alkylene group comprises one to six carbon atoms; (8) azido; (9) cycloalkyl of three to eight carbon atoms; (10) halo; (11) heterocyclyl; (12) (heterocycle)oxy; (13) (heterocycle)oyl; (14) hydroxyl; (15) hydroxyalkyl of one to six carbon atoms; (16) N-protected amino; (17) nitro; (18) oxo or thiooxo; (19) perfluoroalkyl of 1 to 4 carbon atoms; (20) perfluoroalkoxyl of I to 4 carbon atoms; (21) spiroalkyl of three to eight carbon atoms; (22) thioalkoxy of one to six carbon atoms; (23) thiol; (24) A A OC(O)R^, where R is selected from the group consisting of (a) substituted or unsubstituted CI.

6 alkyl, (b) substituted or unsubstituted C 6 or Cio aryl, (c) substituted or unsubstituted C 7

-

16 arylalkyl, where the alkylene group comprises one to six carbon atoms, (d) substituted or unsubstituted C 1

.

9 heterocyclyl, and (e) substituted or unsubstituted C 2

-

15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (25) C(O)RB, where RB is selected from the group consisting of (a) hydrogen, (b) substituted or unsubstituted C 1

.

6 alkyl, (c) substituted or unsubstituted

C

6 or C 1 o aryl, (d) substituted or unsubstituted C 7

-

16 arylalkyl, where the alkylene group comprises one to six carbon atoms, (e) substituted or unsubstituted CI-9 heterocyclyl, and (f) substituted or unsubstituted C 2

-

15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (26) CO 2 RB, where RB is selected from the group consisting of (a) hydrogen, (b) substituted or unsubstituted C 1

.

16 arylalkyl, where the alkylene group comprises one to six carbon atoms, (e) substituted or unsubstituted C1.

9 heterocyclyl, and (f) substituted or unsubstituted C 2 15 heterocyclylalkyl, where the alkylene group comprises one to six carbon atoms; (27) C(O)NRCRD, where each of Rc and RD is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; (28) S(O)RE, where RE is selected from the group consisting of (a) alkyl, (b) aryl, (c) arylalkyl, where the alkylene group comprises one to six carbon atoms, and (d) hydroxyl; (29) S(O) 2 RE, where RE is WO 2012/129673 PCT/CA2012/000316 - 14 selected from the group consisting of (a) alkyl, (b) aryl, (c) arylalkyl, where the alkylene group comprises one to six carbon atoms, and (d) hydroxyl; (30)

S(O)

2 NRF R, where each of RF and RG is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl and (d) arylalkyl, where the alkylene H I H I group comprises one to six carbon atoms; and (31) -NR R , where each of R and R1 is independently selected from the group consisting of (a) hydrogen; (b) an N protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, where the alkylene group comprises one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms, (i) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms, and the alkylene group comprises one to ten carbon atoms, (j) alkanoyl of one to six carbon atoms, (k) aryloyl of 6 to 10 carbon atoms, (1) alkylsulfonyl of one to six carbon atoms, and (m) arylsulfonyl of 6 to 10 carbons atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group. 10045] The term "aryl" as used herein, represents mono- and/or bicyclic carbocyclic ring systems and/or multiple rings fused together and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like and may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, where the alkyl and alkylene groups are independently comprised of one to six carbon atoms; (9) aryl; (10) arylalkyl, where the alkyl group comprises one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, where the alkylene group comprises one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; WO 2012/129673 PCT/CA2012/000316 - 15 (19) (carboxaldehyde)alkyl, where the alkylene group comprises one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms and the alkylene group comprises one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocyclyl; (25) (heterocyclyl)oxy; (26) (heterocyclyl)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, where the alkylene group comprises one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (36) (CH 2 )qCO 2 R^, where q is an integer ranging from zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, where the alkylene group comprises one to six carbon atoms; (37) (CH 2 )qC(O)NR Rc, where RB and Rc are independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; (38) (CH 2 )qS(O) 2 RD, where RD is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, where the alkylene group comprises one to six carbon atoms; (39) (CH 2 )qS(O) 2 NRERF, where each of RE and R is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; (40) (CH 2 )qNRGRH, where each of RG and RH is independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, where the alkylene group comprises one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms, and (i) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms, and the alkylene group comprises one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; (48) arylalkoxy and (49) thiohaloalkyl WO 2012/129673 PCT/CA2012/000316 -16 [0046] The term "aralkyl" represents an aryl group attached to the parent molecular group through an alkyl group. [00471 The term "alkheterocyclyl" represents a heterocyclic group attached to the parent molecular group through an alkyl group. [00481 The term "aryloxy" as used herein, represents an aryl group that is attached to the parent molecular group through an oxygen atom. [0049] The term "alkoxyalkyl" as used herein means alkyl-O-alkyl-, wherein alkyl is defined above. [0050] The term "alkoxyaryl" as used herein means alkyl-O-aryl-, wherein alkyl is defined above. [0051] The term "thioalkyl" as used herein means alkyl-S-, wherein alkyl is defined above. [0052] The term "alkthioalkyl" as used herein means alkyl-S-alkyl-, wherein alkyl is defined above. [00531 The term "alkthioaryl" as used herein means alkyl-S-aryl-, wherein alkyl is defined above. 100541 The terms "aryloyl" or "aroyl" as used interchangeably herein, represent an aryl group that is attached to the parent molecular group through a carbonyl group. [0055] The term "carbonyl" as used herein, represents a C(O) group, which can also be represented as C=O.

WO 2012/129673 PCT/CA2012/000316 -17 [00561 The terms "carboxy" or "carboxyl," as used interchangeably herein, represents a CO 2 H group. [0057] The term "cycloalkyl" as used herein, represents a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of three to eight carbon atoms, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl and the like. The cycloalkyl groups of the present disclosure can be optionally substituted with: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (9) aryl; (10) arylalkyl, where the alkyl group comprises one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, where the alkylene group comprises one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, where the alkylene group comprises one to six carbon atoms; 20) cycloalkyl of three to eight carbon atoms; (21) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms and the alkylene group comprises one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocyclyl; (25) (heterocyclyl)oxy; (26) (heterocyclyl)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N protected aminoalkyl, where the alkylene group comprises one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (36)

(CH

2 )qCO 2 R^, where q is an integer ranging from zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, where the alkylene group comprises one to six carbon atoms; (37) (CH 2 )qC(O)NR BRc, where each of RB and RC WO 2012/129673 PCT/CA2012/000316 -18 is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl, where the alkylene group comprises one to six carbon atoms; (38) (CH 2 )qS(O) 2 RD, where RD is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, where the alkylene group comprises one to six carbon atoms; E F (39) (CH 2 )qS(O) 2 NR R , where each of RE and RF is independently, selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl, where the G H alkylene group comprises one to six carbon atoms; (40) (CH 2 )qNR R , where each of RG and R is independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, where the alkylene group comprises one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms and (i) alkcycloalkyl, where the cycloalkyl group comprises three to eight carbon atoms, and the alkylene group comprises one to ten carbon atoms, with the proviso that no two groups are bound to the nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy. [0058] The term "halogen" or "halo" as used interchangeably herein, represents F, Cl, Br and I. [00591 The term "heteroaryl" as used herein, represents that subset of heterocycles, as defined herein, which is aromatic: (i.e., containing 4n+2 pi electrons within a mono- or multicyclic ring system). [00601 The terms "heterocycle" or "heterocyclyl" as used interchangeably herein represent a 5-, 6- or 7-membered ring, unless otherwise specified, comprising one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The 5-membered ring has from zero to two double bonds and the 6- and 7-membered rings have from zero to three double bonds. The term "heterocycle" also includes bicyclic, tricyclic, and tetracyclic groups in which WO 2012/129673 PCT/CA2012/000316 - 19 any of the above heterocyclic rings is fused to one or two rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring and another monocyclic heterocyclic ring such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Heterocycles include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, isoindazoyl, triazolyl, tetrazolyl, oxadiazolyl, uricyl, thiadiazolyl, pyrimidyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroinidolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, benzothienyl and the like. Heterocyclic groups also include compounds of the formula ~ F~ /G' [0061] I o' , where F' is selected from the group consisting of

CH

2 , CH 2 0 and 0, and G' is selected from the group consisting of C(O) and (C(R')(R")),, where each of R' and R" is independently selected from the group consisting of hydrogen and alkyl of one to four carbon atoms, and v is an integer ranging from one to three, and includes groups such as 1,3-benzodioxolyl, 1,4 benzodioxanyl and the like. Any of the heterocyclic groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, where the alkyl and alkylene groups independently comprise from one to six carbon atoms; (9) WO 2012/129673 PCT/CA2012/000316 - 20 aryl; (10) arylalkyl, where the alkyl group comprises one to six carbon atoms; (11) amino; (12) aminoalkyl of one to six carbon atoms; (13) aryl; (14) arylalkyl, where the alkylene group comprises one to six carbon atoms; (15) aryloyl; (16) azido; (17) azidoalkyl of one to six carbon atoms; (18) carboxaldehyde; (19) (carboxaldehyde)alkyl, where the alkylene group comprises one to six carbon atoms; (20) cycloalkyl of three to eight carbon atoms; (21) alkcycloalkyl, where the cycloalkyl group comprises from three to eight carbon atoms and the alkylene group comprises from one to ten carbon atoms; (22) halo; (23) haloalkyl of one to six carbon atoms; (24) heterocycle; (25) (heterocycle)oxy; (26) (heterocycle)oyl; (27) hydroxy; (28) hydroxyalkyl of one to six carbon atoms; (29) nitro; (30) nitroalkyl of one to six carbon atoms; (31) N-protected amino; (32) N-protected aminoalkyl, where the alkylene group comprises from one to six carbon atoms; (33) oxo; (34) thioalkoxy of one to six carbon atoms; (35) thioalkoxyalkyl, where the alkyl and alkylene groups AA independently comprise from one to six carbon atoms; (36) (CH2)qCO2RA, where q is an integer ranging from zero to four and RA is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, where the alkylene group comprises from one to six carbon atoms; (37) (CH 2 )qC(O)NRBRc, where each of RB and RC is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl, where the alkylene group comprises from one to six carbon atoms; (38)

(CH

2 )qS(O) 2 R D, where RD is selected from the group consisting of (a) alkyl, (b) aryl, and (c) arylalkyl, where the alkylene group comprises from one to six carbon atoms; E FE (39) (CH 2 )qS(O) 2 NR R , where each of RE and RF is independently selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) arylalkyl, where the alkylene group comprises from one to six carbon atoms; (40) (CH 2 )qNRGRH, where each of RG and RH is independently selected from the group consisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g) arylalkyl, where the alkylene group comprises from one to six carbon atoms; (h) cycloalkyl of three to eight carbon atoms, and (i) alkcycloalkyl, where the cycloalkyl group comprises from three to eight carbon atoms, and the alkylene group comprises from one to ten carbon atoms, with the proviso that no two groups are bound to the WO 2012/129673 PCT/CA2012/000316 -21 nitrogen atom through a carbonyl group or a sulfonyl group; (41) oxo; (42) thiol; (43) perfluoroalkyl; (44) perfluoroalkoxy; (45) aryloxy; (46) cycloalkoxy; (47) cycloalkylalkoxy; and (48) arylalkoxy. [00621 The terms "heterocyclyloxy" or "(heterocycle)oxy" as used interchangeably herein, represents a heterocyclic group, as defined herein, attached to the parent molecular group through an oxygen atom. [00631 The term "heterocyclyloyl" or "(heterocycle)oyl" as used interchangeably herein, represents a heterocyclic group, as defined herein, attached to the parent molecular group through a carbonyl group. 10064] The term "amino acid", as used herein, is understood as including both the L and D isomers of the naturally occurring amino acids, as well as other non proteinaceous amino acids used in peptide chemistry to prepare synthetic analogs of peptides. Examples of naturally-occurring amino acids include, but are not limited to glycine, alanine, valine, leucine, isoleucine, serine, and threonine. Examples of non proteinaceous amino acids include, but are not limited to norleucine, norvaline, cyclohexyl alanine, biphenyl alanine, homophenyl alanine, naphthyl alanine, pyridyl alanine, and substituted phenyl alanines (substituted with a or more substituents including but not limited to alkoxy, halogen and nitro groups). Beta and gamma amino acids are also within the scope of the term "amino acid". Amino acids protected by standard protecting groups commonly used in peptide synthesis are also within the scope of the term "amino acid". These compounds are known to persons skilled in the art of peptide chemistry. [0065] The term "oxo" as used herein, represents =0. [00661 The term "perfluoroalkyl" as used herein, represents an alkyl group, as defined herein, where each hydrogen radical bound to the alkyl group has WO 2012/129673 PCT/CA2012/000316 - 22 been replaced by a fluoride radical. Perfluoroalkyl groups are exemplified by trifluoromethyl, pentafluoroethyl, and the like. [0067] The term "heteroatom", as used herein, is understood as being oxygen, sulfur or nitrogen. [0068] The term "sulfonyl" as used herein, represents an S(O) 2 group. [00691 The term "thioalkoxy" as used herein, represents an alkyl group attached to the parent molecular group through a sulfur atom. Exemplary unsubstituted thioalkoxy groups comprise from I to 6 carbon atoms. 100701 The term "thiocarbonyl" as used herein, represents a C(S) group, which can also be represented as C=S. [0071] The term "pharmaceutically acceptable salt," as used herein, refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. The salts can be prepared in situ during the final isolation of the compounds, or separately by reacting the free base or acid function with a suitable organic acid or base, respectively. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, WO 2012/129673 PCT/CA2012/000316 -23 pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxy group (or other acidic moiety) with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine. The cations of pharmaceutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, I-ephenamine, and N,N'-dibenzylethylenediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine and piperazine. 100721 170-HSDI [00731 To obtain a further interaction (H-bonding or hydrophobic interaction) in the binding pocket in proximity of position 3 of I, and therefore improve its binding with 17p-HSDI, various functional groups as well as various changes in the length of the side chain were explored (Scheme 1). Moreover, it is widely surmised that replacing the OH at position C3 could remove the undesirable estrogenic activity by disturbing the binding to ER. In fact, a phenolic OH appears to be a crucial requirement for ER binding.

38 Based on SAR (Structure Activity Relationship) studies, numerous C3 derivatives of I were prepared and tested, examples of which include compounds II-V (Scheme 1). It was surmised that this modification at C3 would result in compounds having an additional interaction in the binding pocket of 17p-HSDI and hence improve their binding while concomitantly eliminating the undesirable estrogenic activity observed for I.

WO 2012/129673 PCT/CA2012/000316 - 24 [00741 Compounds II and III were synthesized from 3-carboxy-estrone.

39 The benzylcarbamide side chain was introduced at position C16 by means of an aldol condensation reaction with 3-formyl benzamide.

4 0

,

41 The C 17 ketone was then reduced using sodium borohydride and the 16-exo double bound was subsequently reduced using hydrogenation with Pd (10%) on charcoal (10%). The 3-carboxylic acid was then activated using BOP to promote reduction using sodium borohydride to provide the corresponding alcohol II. Alcohol II was subsequently brominated using triphenylphosphine and carbon tetrabromide to provide the bromomethyl bromide derivative III (Scheme 2). [00751 Compounds IV and V were synthesized from 3-vinyl-17 dioxolane-estrone (1) which is readily obtained from carbonylative vinylation of estrone triflate followed by C17 dioxolane protection.42 The vinyl group was first converted into the primary alcohol 2 by oxidative hydroboration using BH 3

-DMS.

43 Alcohol 2 was subsequently protected as the benzyl ether followed by dioxolane deprotection in acidic conditions to provide 3. The benzylcarbamide side chain was introduced at position C16 by means of an aldol condensation reaction with 3-formyl benzamide. The C 17 ketone was then reduced using sodium borohydride and the 16 exo double bound was subsequently reduced using hydrogenation with Pd (10%) on charcoal (10%). The hydrogenation reaction concomitantly cleaved 3-0-benzylether to provide the m-16p-carbamoylbenzyl derivative IV. Alcohol IV was subsequently brominated using triphenylphosphine and carbon tetrabromide to provide the bromoethyl derivative V (Scheme 2).

WO 2012/129673 PCT/CA2012/000316 -25 O 0 OH a H 0 b, c H d X ONH2 OH 11 (X ~OH) III(X =VB() 0 0 H H g, h H 12 3 0 OH a H - 0 b, cH0 BnO' NI1 X -H

NH-

2 4 x IV(X =OH) V (X = Br) Scheme 2: Reagents and conditions: (a) 3-carboxamide-benzaldehyde, KOH, EtOH, rt; (b) NaBH 4 , MeOH; (c) H 2 , Pd/C, MeOH; (d) 1) BOP, DIPEA, DMF, rt; 2) NaBH 4 , MeOH; (e) PPh 3 , CBr 4 , DCM, rt; (f) 1) BH 3 -DMS, THF, -78-C; 2) H 2 0 2 , NaHCO 3 ; (g) NaH, Benzylbromide, DMF; (h) 10% HCL: acetone (1:1). 100761 Additional C3 derivatives of I were prepared as illustrated herein below in Schemes 3-14.

WO 2012/129673 PCT/CA2012/000316 -26 OH OH 0 0 HO

NH

2

NH

2 7 8 kI OH OH 0 0O C1

NH

2

ONH

2 9 10 OH 0

NH

2 Reagents and conditions: (i) PPh 3 , CBr 4 , DCM, rt; (j) CPMA, DCM Scheme 3 OH OH H a N HO

-

0 0

NH

2 13 NH 2 12 OH OH HH C HO, OCBr O 0 0 Br,,-0 0 14 NH 2 16 NH 2 Reagents and conditions: (a) allytbronide, NaOH, acetone, rc; (b) i) NatO 4 RuC13 X H 2 0, EtOAc/ACN; ii) NaBH 4

H

2 0; (c) PPh 2 CBr 4 DCM, 0 to rt. Scheme 4 WO 2012/129673 PCT/CA2012/000316 -27 0 O H a b~c H O N. I H H 0. NH2 N. 0OO O NH 2 19 Reagents and conditions: (a) styrene, Grubb (11) catalyst; Dichiroethane, rx; (b) HCI 10% in MeOH, rt; (c) 3-carboxamide-benzaldehyde, KOH,EtOH, rx; (d) NaBH4 MeOH; () H2 Pd/C, EtOH, rt Scheme 5 O a BnO O bBnO. 120 21 H 0 H C Bn NH2 d ,Hn NH2 22 23 OH H OH e , O NH2 BH NH2 24 25 Reagents and conditions: (a) Grubb 11 catalyst, allyloxymethyl-benzene; (b) HCI, MeOH; (c) 3-carboxamide-benzaldehyde, KOH, EtOH, rx; (d) NaBH4 MeOH; ( a) H 2 Pd/C, MeOH: (f PPh, CBr4 DCM, rt. Scheme 6 WO 2012/129673 PCT/CA2012/000316 -28 0 ~ 00 a b 1 26 27 NH 2 OH OH c H d H 28 NH 2 29 NH 2 Reagents and conditions: (a) HCI 10% in MeOH, rt; (b) 3-carboxamide-benzaldehyde, KOH,EtOH, rx; (d) NaBH 4 MeOH; (e) H 2 Pd/C, EtOH, rt Scheme 7 OH 0 N a H0 HO ONH 2 O0 NH 2 HO HOJ 11 30 OH 4c,b b H 0 OH 0 0 I Ft Ht NH 2 H HO H2 NH 2 31 H 32 Reagents and conditions: (a) Dess-Martin, NaOC 2 t-BuOH, 2-methyl-butene, KH 2

PO

4 (b) NaBH 4 MeOH: (c) BOP, DIPEA, Methylamine in THF. Scheme 8 WO 2012/129673 PCT/CA2012/000316 -29 0H O HOO OHH BrNH HO 2b gHO = - HON 0 H 0 NH 2 NH 29N 2 339 OH OH H R C 8 0 H NR__ _ )__ H) HON HOON R == ---N ; -N ; H o 36 0- NH 37 N OH40 40 40 40 HO H2N 30

NH

2 Reagents and conditions: (a)-ro md R1R NH, BOP , PEA H K b)NBHDM 4eHD M(:) ()H.P/ 10) 41bH 41C 41dPDPATF S eme 0 ( ; H H NH HO H 0 NH0N Reagents and conditions: (a) 2-eoaieboadhde O,0Ox()NB 2 .MO/C 11;()H P/ 1%.EO;() BOP, DIPEA, THF:a 1 4c 1 Scheme 90 WO 2012/129673 PCT/CA2012/000316 -30 0 0 a H b H, 0,00 HO,I TfO 0 HO 0 42 43 44 NH 2 OH OH c H ~ dH N -p d - N HO, H H HO, | H H HO 0 HO O 45 NH 2 46 NH2 Reagents and conditions: (a) Pinacolborane, Pd(dppf)C 2 , DCM, dioxane, 100*C; (b) 3-carboxamide benzaldehyde, KOH, EtOH, rx; (c) NaBH 4 , MeOH; (d) H 2 , Pd/C (10%), EtOH. Scheme 11 0 0 aH H 2 N

H

2 N 47 0 NH2 OH OH b H -- C H2N R 1 , N 49 0 H 0

NH

2 NH 2 52 53 dI OH R1,N N H 0 NH2 51 Reagents and conditions: (a) 3-carboxamide-benzaldehyde, KOH, EtOH, rx; (b) NaBH4, MeOH, rt; (c) Pd/C (10%), MeOH; (d) Pd/C (10%), EtOH, rt. Scheme 12 WO 2012/129673 PCT/CA2012/000316 -31 00 O a b H H 2 N' H. H 4754 55

NH

2 c OH OH d H H F F 01 O NH 2 57 0 NH 2 Reagents and conditions: (a ) t-buty nitrite, BF 3 0(Et) 2 . DCM; (b) 3-carboxamide-benzaldehyde, KOH, EtOH, rx; (c) NaBH 4 .MeOH; (d) H 2 , Pd/C (10%), EtOH Scheme 13 00 OH H2N H H2N b H2N HN

H

2 2NN H2 2 N N 0 80 5 0 NH 2 0 60 0NH 2 OH CR

H

2 N NN 61 0 NH 2 Reagents and conditions: (a) 3-carboxamide-benzaldehyde, KOH, EtCH, ma; (bi) NaBH 4 , MeCH/DCM (9:1), it: (c) Pd/C (10%), EtCH, rt. Scheme 14 100771 In one embodiment of the present disclosure, there are included inhibitors of I 7B-HSDI, in which the inhibitor has the structure: WO 2012/129673 PCT/CA2012/000316 - 32 wherein R is -OH, halo, -NH 2 , alkyl, alkenyl, alkoxy, aralkyl, carboxy, -CH 2 heterocyclyl, heterocyclyloyl, -C(O)N(R')(R") or -B(OH) 2 , wherein R' and R" are independently or simultanesouly H or alkyl, or R' and R" are joined together, along with the nitrogen atom to which they are attached, to form a heterocyclic ring, or a pharaceutically acceptable salt or tautomer thereof. 100781 In one embodiment, the 17B-HSDI inhibitor is the compound V, or a pharmaceutically acceptable salt thereof. In one embodiment, the inhibitor is the corresponding hydrochloride (HCI) salt of compound V, or other acid addition salt on the carboxamide moiety of compound V. 100791 In another embodiment, the 17B-HSD1 inhibitor is a radiolabelled derivative of compound V having the structure OH x 0 NH2, wherein X is m 2 131 or Br76 [00801 In another embodiment, the 17B-HSDI inhibitor is a radiolabelled derivative of compound V having the structure OH

X

WO 2012/129673 PCT/CA2012/000316 - 33 wherein X is 1123 1125, 131 or Br 76. 123 125 131 [0081] In another embodiment, the radiolabel is located (e.g. I, 12, 1 or Br 76 ) in a suitable positions on the steroid core of compound V providing for additional radiolabelled derivatives. [0082] The radiolabelled derivatives of the compound V as shown above are useful for the radioimaging and radiotherapeutic treatment of breast cancer and prostate cancer, as such cancers are known to express 17B-HSD, in addition to any other cancers which express 17B-HSD such as endometrial cancer. 100831 In another embodiment, the 17B-HSD1 inhibitor is the alpha acetylenic derivative of the compound V having the following structure OH H H H NH2 Br [0084] Biological Activity 100851 Compounds I-V were tested for their ability to inhibit the transformation of El into E2 by 17p-HSD1 in T-47D cells (Table 1), a cell line well known to express endogeneous 17p-HSDI. The effect of the inhibitors on the growth of estrogen-starved estrogen sensitive MCF-7 cells was also evaluated (Table 1). In the alcohol series, it was observed that extending the hydroxyl away from ring A of the steroid structure resulted in a negative effect on the inhibition potency, with values dropping from 66% for phenol I to 37% for methyl alcohol II and 17% for ethyl WO 2012/129673 PCT/CA2012/000316 - 34 alcohol IV when tested at 0.1 pM. These results, and those obtained at lower concentrations (0.01 pM), highlight the fact that no additional interaction was obtained in the binding pocket of 17p-HSDl. The bromoalkyl derivatives III and V proved more promising. In fact, the inhibitory activity of V (IC 50 = 68 nM) was just two times lower than that of reference inhibitor I (IC 50 = 27 nM). More importantly, the estrogenic activity was greatly modulated by the presence of the C3-side chain (FIG. 1). Compound I, bearing a hydroxyl at position C3, strongly stimulated the estrogen sensitive MCF-7 cells at concentration ranging from 0.1 pM to 5 pM. Extending the hydroxyl away from ring A of the steroid structure (compound II) greatly decreased the estrogenicity of the compound while maintaining significant estrogenic activity at doses ranging from 0.5 mM to 5 pM. Extending the hydroxyl by a further methylene unit (compound IV) completely removed any estrogenic activity. The same observations were made with the corresponding bromide (compound V) showing no estrogenicity at any of concentrations ranging from 0.5 pM to 5 pM. 10086] Table 1: Inhibition Values and Estrogenic Activities for Compounds I-V OH H 0 NH 2 Compound R Inhibition % inhibition a IC 50 __________________ (0.01 PNM)a (0. 1 AM~)' (nMI)b I OH 35 66 27±4 II CH 2 OH 14 37 N/A III CH 2 Br 20 36 N/A IV CH 2

CH

2 OH 7 17 N/A V CH 2

CH

2 Br 23 49 68±6 "Inhibition of the transformation of [ 14 C]-El (60 nM) into [ 14 C]-E2 by 17p-HSDI in T-47D intact cells. The experiments were performed in triplicate (SD < ± 5%). bMean ± SD of an experiment performed in triplicate.

WO 2012/129673 PCT/CA2012/000316 -35 [00871 Compound V proved to be the most active inhibitor, demonstrating the tolerance of 17p-HSDI for a hydrophobic chain introduced at position C3. Most importantly, the substitution of the hydroxyl group of I with the hydrophobic chain of V, proved to be very efficient for removing any residual estrogenic activity while undergoing only a slight drop in inhibitory activity. In view of the very good inhibitory activity of V (ICso = 68 nM) and more importantly in view of the absence of any estrogenic effect observed on estrogen-sensitive breast cancer cells, V represents an excellent candidate for in vivo studies targeting 17p-HSD1. [00881 Compound V was also tested with estradiol in a competitive binding assay on a human estrogen receptor alpha. FIG 2. shows the competitive binding in which Compound V binds to the receptor at all concentrations. [0089] Compound V was also tested on the 17p-HSD2 enzyme for the conversion of E2 to El at the tested concentrations varying from 5 nM to 20 pM, and demonstrated no inhibition for this enzyme. [00901 Compound V was also tested on CYP3A4 and demonstrated an

IC

50 of 4,06 + 0,57 pM, compared to 1,52 ± 0,29 pM for compound I. [00911 Compounds 7-10, 15, 19, 24, 25, 29, 31, 32, 36-41, 46, 50, 57 and 61 were tested for their ability to inhibit the transformation of El into E2 by 17p HSDI in T-47D cells (Table 2), a cell line well known to express endogeneous 17p HSD1. The effect of the inhibitors on the growth of estrogen-starved estrogen sensitive MCF-7 cells was also evaluated (Table 2). [0092] Table 2: Inhibition Values and Estrogenic Activities for Compounds 7-10, 15, 19, 24, 25, 29, 31, 32, 36-41, 46, 50, 57 and 61.

WO 2012/129673 PCT/CA2012/000316 -36 OH R 0

NH

2 Compound R Inhibitiona Inhibitiona Estrogenicityb Estrogenicityb # (0.1 PM) (10 4M) (0.1 tM) (1.0 p.M) DP-156 --OH 75 90 ++ ++ 7 OH 19 57 + ++ 8 Br 56 89 9 55 N/A N/A N/A 10 ci 7 N/A N/A N/A 15 39 81 N/A N/A 19 13 N/A N/A N/A 24 OH 16 N/A N/A N/A 25 Br 37 82 N/A N/A 29 12 N/A N/A N/A 0 31 >-kOH 12 29 - + 0 32 N 5 15 H 36 OH 5 8 - ++ 0 OH 37 OH 56 73 - + 38 Br 19 66 39 NH 2 35 73 ++ ++ 40a N / 12 49 40b 5 27 40c N3 1 28 - WO 2012/129673 PCT/CA2012/000316 -37 40d H 26 65 H N 41d N 3 5 - + 0 41a 2 16 + + 0 41b N 1 14 0 41c N~ 7 I 0 13 0 46 -_ -PH 39 N/A ++ ++ OH 50 --NH 2 35 43 - + 57 --F 48 50 - + 61 NH 2 3 23 - ++ 0 "Inhibition of the transformation of [ 4 C]-El (60 nM) into [ 14 C]-E2) by 17p-HSD1 in T-47D intact cells. The experiment was performed in triplicate (SD < ± 5%). The inhibitors were tested at two concentrations of 0.1 and 1 pM. bEffect of inhibitors on the growth of estrogen starved estrogen sensitive MCF-7 cells after 7 days of treatment at different concentrations. Legend: - = no; + = weak ++ = medium to strong. [0093] EXPERIMENTAL [00941 Materials, Methods, Synthesis and Characterization [0095] Chemical reagents were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). The usual solvents were obtained from Fisher Scientific (Montr6al, QC, Canada) and were used as received. Anhydrous dichloromethane (DCM), dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained from Sigma-Aldrich. Thin-layer chromatography (TLC) and flash-column chromatography were performed on 0.20-mm silica gel 60 F254 plates and with 230-400 mesh ASTM silica gel 60, respectively (E. Merck; Darmstadt, Germany). Infrared spectra (IR) were recorded using a Horizon MB 3000 ABB FT-IR spectrometer and the significant WO 2012/129673 PCT/CA2012/000316 - 38 bands reported in cm- 1 . Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for 1 H and 100.6 MHz for 1 3 C using a Bruker Avance 400 digital spectrometer (Billerica, MA, USA). The chemical shifts (6) were expressed in ppm and referenced to chloroform (7.26 and 77.0 ppm), acetone (2.06 and 29.24 ppm) or methanol (3.31 ppm and 49.0) for 1 H and 13 C NMR respectively. Low-resolution mass spectra (LRMS) were recorded using a PE Sciex API-150ex apparatus (Foster City, CA, USA) equipped with a turbo ion-spray source and expressed in m/z. High resolution mass spectra (HRMS) were provided by Pierre Audet at the Ddpartement de Chimie de l'Universit6 Laval (Quebec, QC, Canada). The purity of the compounds was determined by high-performance liquid chromatography (HPLC) (Waters Associates Milford, MA, USA) using a Luna phenyl hexyl column (75 x 4.6 mm id, 3 tm, serial N 0 : 338048-2, 60A) or a Nova Pak C 18 reverse-phase column (150 mm x 3.0 mm id, 4 pm, 60A). 100961 Cell Culture 10097] The ER-positive breast cancer cell lines T-47D and MCF-7 cells were obtained from the American Type Culture Collection (ATCC) and maintained in 175 cm2 culture flasks at 37'C in a humidified atmosphere at 5% CO 2 . The T-47D cells were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 pg/mL) and estradiol (1 nM). The MCF-7 cells were propagated in Dubelcco's Modified Eagle Medium: Nutrient Mixture F-12 Ham (DMEM-F12) medium supplemented with 5% FBS, glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 [tg/mL) and estradiol (1 nM). [00981 Inhibition of 179-HSD1 in T-4 7D Cells [0099] T-47D cells were seeded in a 24-well plate (3000 cellsvell) in medium supplemented with insulin (50 ng/mL) and 5% dextran-coated charcoal WO 2012/129673 PCT/CA2012/000316 - 39 treated FBS rather than 10% FBS to remove remaining hormones. After 24 h, the cells were incubated with 60 nM of [ 14 C]-Estrone (American Radiolabeled Chemicals, Inc., St.Louis, MO, U.S.A). An ethanolic solution of inhibitors (0.5 %vv) at concentrations of 0.01 tM and 0.1 pM for all inhibitors were added to freshly changed cultured medium and the cells were incubated over a period of 24 h. For the most active inhibitors, concentrations ranging from 0.01 pM to 10 pM were tested to determinate their IC 50 values. Each inhibitor was assessed in triplicate. After incubation, the culture medium was removed and labelled steroids (El and E2) extracted with I mL of diethyl ether. The organic phases were separated and evaporated to dryness using nitrogen. Residues were dissolved in dichloromethane and dropped on silica gel thin layer chromatography plates (EMD Chemicals Inc., Gibbstown, NJ, USA) and eluted with a toluene/acetone (4:1) solvent system. Substrate [ 14 C]-El and metabolite [ 14

C]

E2 were identified by comparison with reference steroids and quantified using a Storm 860 system (Molecular Dynamics, Sunnyvale, CA, USA). The percentage of transformation and inhibition were calculated as follows: %transformation= 100 x

['

4 C]-E2/([' 4 C]-El+ [ 4 C]-E2) and % of inhibition = 100 x (%transformation without inhibitor-%transformation with inhibitor)/ %transformation without inhibitor. 100100] Estrogenic Activity in MCF- 7 Cells [001011 MCF-7 cells were seeded with medium supplemented with insulin (50 ng/mnL) and 5% dextran-coated charcoal treated FBS rather than 10% FBS to remove remaining hormones. Aliquots (100 pL) of the cell suspension were seeded in 96-well plates (3000 cells/vell). After 48 h, the medium was replaced with a fresh one containing an appropriate concentration of products to be tested and was replaced every 2 days. Cells were grown either in the absence or presence of the compounds over a period of 7 days. Quantification of cell growth was determined by using the CellTiter 96@Aqueous Solution Cell Proliferation Assay (Promega, Nepean, ON, Canada) following the manufacturer's instructions. To determine the proliferative (estrogenic) activity, the cells were grown in the absence (control fixed as 100%) or presence of the tested compounds at the indicated concentrations.

WO 2012/129673 PCT/CA2012/000316 - 40 1001021 ER alpha binding assay with Compound V [00103] A competitive binding assay using a purified full-length recombinant human ERa (Life Technologies, Grand Island, NY) was done as previously described (Davis et al. J. Steroid Biochem. Mol. Biol., 2008, 108, 23-31; Arcaro et al. J Cell. Biochem. 1999, 72, 94-102). Briefly, each reaction consisted of 1.2 nM rhERa and 2.5 nM [ 3 H]-estradiol in assay buffer (10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, I mg/mL BSA, pH 7.5) with different concentrations of the compounds or cold estradiol in a total reaction volume of 100 ptL. Non-specific binding was determined by incubation with an excess of cold estradiol (1 1 M). After an overnight incubation at 4'C, 100 pl of cold 50% hydroxyapatite slurry was added to bind the receptor/ligand complex. After 15 minutes, 1 mL of wash buffer (40 mM Tris, 1 mM EDTA, 1 mM EGTA, 100 mM KCl, pH 7.4) was added and the tubes were centrifuged at 4500 rpm for 5 minutes at 4'C. The washing step was repeated twice. The radioactivity of the pellet was extracted by incubation with I mL of ethanol for I h at room temperature. The suspension was then put into 10 mL of Biodegradable Counting Scintillant and the radioactivity counted with a Wallac 1411 Liquid Scintillation Counter. IC 50 values were obtained using GraphPad Prism 5 and RBA values were obtained by using the following equation: (IC 50 of 171-estradiol / IC 50 of compound) x 100. ICso RBA Estradiol 1.02 nM 0.09 nM 100 Compound V- [00104] Inhibition of 1 70-HSD2 with Compound V [001051 Inhibition assays were performed using stable 17p-HSD2 HEK 293 transfected intact cells with 5000 cells per well using 24 well plates (Poirier et al. Mol. Cell. Endocrinol., 2001, 171, 119-128). The cells were incubated for 1 h at 37'C in I mL of MEM medium steroid free with 60 nM C 14 estradiol. The steroids were WO 2012/129673 PCT/CA2012/000316 -41 then extracted, quantified and separated as previously described for 17p-HSDI enzymatic activity and the % inhibition calculated (Tremblay et al J. Enzyme Inhib. Med. Chem., 2005, 20, 153-163). Compound V did not show any inhibition on the 1 70-HSD2 enzyme for the conversion of E2 to E I at the tested concentrations varying from 5 nM to 20 pM. [001061 Inhibition of CYP3A4 with Compound V [001071 The P450 Inhibition Kit CYP3A4/DBF purchased from BD Biosciences was used as suggested by the company, with the exception that the compound V was dissolved in a mixture of 5%DMSO/95%acetonitrile 100108] Compound V demonstrated a lower inhibition of CYP3A4 with a IC50 of 4.06 ± 0.57 pM, compared to 1.52 ± 0.29 pM for compound 1. [001091 17p-HSD1 Inhibition in Breast Cancer Cell Lines with Compound V 100110] In vitro studies-Cell culture [001111 Breast cancer cell line T-47D was obtained from the American Type Culture Collection (ATCC) and maintained in a 175 cm 2 culture flask at 37 'C in a humidified atmosphere at 5% CO 2 . Cells were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 pg/mL) and estradiol (1 nM). [00112] 17p-HSD1 Inhibition Assay 1001131 T-47D cells were seeded in a 24-well plate (3000 cells/well) in 990 pL of medium supplemented with insulin (50 ng/mL) and 5% dextran-coated WO 2012/129673 PCT/CA2012/000316 - 42 charcoal-treated FBS, which was used rather than untreated 10% FBS, to remove the remaining steroid hormones. Stock solutions of inhibitors Compounds I and V were previously prepared in ethanol and diluted with culture medium to achieve appropriate concentrations prior to use. After 24 h of incubation, 5 IL of the diluted solution were added to the cells to obtain a final concentration ranging from I nM to 10 ptM to determine the IC 50 value. The final concentration of ethanol in the well was adjusted to 0.1%. Additionally, 5 pL of a solution of [ 14 C]-estrone (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) was added to obtain a final concentration of 60 nM. Cells were incubated for 24 h and each inhibitor was assessed in triplicate. After incubation, the culture medium was removed and labeled steroids (El and E2) were extracted with I mL of diethyl ether. The organic phases were evaporated to dryness with nitrogen. Residues were dissolved in dichloromethane and dropped on silica gel thin layer chromatography plates (EMD Chemicals Inc., Gibbstown, NJ, USA) and eluted with toluene/acetone (4:1) as solvent system. Substrate [ 14 C]-E1 and metabolite [ 14 C]-E2 were identified by comparison with reference steroids (El and E2) and quantified using the Storm 860 system (Molecular Dynamics, Sunnyvale, CA, USA). The percentage of transformation and the percentage of inhibition were calculated as follow: % transformation = 100 x [4C]-E2 / ([ 4 C]-El + [' 4 C]-E2) and % of inhibition = 100 x (% transformation without inhibitor - % transformation with inhibitor) / % transformation without inhibitor (Tremblay MR, Boivin RP, Luu-The V, Poirier D. Inhibitors of type I 17beta-hydroxysteroid dehydrogenase with reduced estrogenic activity: modifications of the positions 3 and 6 of estradiol. J Enzyme Inhib Med Chem 2005;20:153-63; Cadot C, Laplante Y, Kamal F, Luu-The V, Poirier D. C6-(N,N-butyl-methyl heptanamide) derivatives of estrone and estradiol as inhibitors of type I 17beta hydroxysteroid dehydrogenase: Chemical synthesis and biological evaluation. Bioorg Med Chem 2007;15:714-2). 100114] Cell Proliferation Assays (170-HSD1 inhibitory, estrogenic and antiestrogenic activities) WO 2012/129673 PCT/CA2012/000316 - 43 1001151 Quantification of cell growth was determined by using CellTiter 96@Aqueous Solution Cell Proliferation Assay (Promega, Nepean, ON, Canada) following the manufacturer's instructions. T-47D cells were resuspended with the medium supplemented with insulin (50 nginL) and 5% dextran-coated charcoal treated FBS rather than 10% FBS to remove remaining hormones. Aliquots (100 PL) of the cell suspension were seeded in 96-well plates (3000 cells/well). After 48 h, the medium was changed with a new one containing an appropriate concentration of products to be tested and was replaced every 2 days. Cells have grown either in absence or presence of the compounds for 7 days. To determine the proliferative (estrogenic) activity, the estrogen-sensitive T-47D cells were grown in absence (basal cell proliferation was fixed as 100%) or presence of compounds to be tested at 0.5 to 10 pM. The potent estrogen E2 was used as a reference control. To determine the inhibition of El-induced cell proliferation, the T-47D cells were grown in the presence of El (0.1 nM) without (control) or with the inhibitor at a concentration of 0.5, 1, 2.5, and 5 pM. The cell proliferation without El ± inhibitor (control) was fixed as 100%. To determine the potential antiestrogenic activity of inhibitor Compound V, the T-47D (ER t ) cells were grown in the presence of estrogen E2 (0.1 nM) and pure antiestrogen EM-139 (0.5 pM) [34] or inhibitor Compound V (0.5 [M). The cell proliferation without E2 and tested compounds (control) were fixed as 100%. [001161 ERa Binding Assay 100117] A competitive binding assay using a purified full-length recombinant human ERa (Life Technologies, Grand Island, NY). Briefly, each reaction consisted of 1.2 nM rhERa and 2.5 nM [ 3 H]-estradiol in assay buffer (10 mM Tris, 1.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, I mg/mL BSA, pH 7.5) with different concentrations of the compounds or untritiated estradiol (E2) in a total reaction volume of 100 L. Non-specific binding was determined by incubation with an excess of E2 (I RM). After an overnight incubation at 4 OC, 100 pL of cold 50% hydroxyapatite slurry was added to bind the receptor/ligand complex. After 15 minutes, I mL of wash buffer (40 mM Tris, 1 mM EDTA, 1 mM EGTA, 100 mM WO 2012/129673 PCT/CA2012/000316 - 44 KCI, pH 7.4) was added and the tubes were centrifuged at 4500 rpm for 5 minutes at 4 'C. The washing step was repeated twice. The radioactivity of the pellet was extracted by incubation with I mL of ethanol for I h at room temperature. The suspension was then put into 10 mL of Biodegradable Counting Scintillant and the radioactivity counted with a Wallac 1411 Liquid Scintillation Counter. IC 50 values were obtained using GraphPad Prism 5 and RBA values were obtained by using the following equation: (IC 50 of 17P-E2 / IC 50 of tested compound) x 100. 1001181 In vivo Studies-Animals [001191 All animals were acclimatized to environmental conditions (temperature: 22 ± 3 'C; humidity: 50 ± 20%; 12-h light/12-h dark cycles, lights on at 07:15 h) for at least 3 days before starting the experiment. The animals were allowed free access to water and a certified commercial rodent food (Rodent Diet #T.2018.15, Harlan Teklad, Madison, WI, USA) and randomized according to their body weight. The experiments with animals were conducted in an animal facility approved by the Canadian Council on Animal Care (CCAC) and the Association for Assessment and Accreditation of Laboratory Animal Care. The study was performed in accordance with the CCAC Guide for Care and Use of Experimental Animals. Institutional approval was obtained. [001201 Plasmatic Concentration of Inhibitor after a Single Subcutaneous Injection 1001211 Six week-old male Sprague-Dawley rats (Crl:CD®(SD)Br VAF/Plus

T

m) weighing approximately 220 g were obtained from Charles-River, Inc. (St-Constant, Qc., Canada). The animals were housed 3 per cage. A pharmacokinetic study was carried out following one subcutaneous (s.c.) injection of the inhibitor at one concentration (2.3 mg/kg of body weight in 0.5 mL of vehicle fluid). The inhibitor was first dissolved in ethanol (EtOH) and thereafter we added propylene WO 2012/129673 PCT/CA2012/000316 - 45 glycol (PG) to obtain a final concentration of EtOH of 8%. During this experiment, the rats were housed individually and were fasted for 8 h before inhibitor injection but allowed free access to water. Blood samples for determination of inhibitor plasma concentration were collected at the jugular vein (0.4 mL by animal) at target intervals of 3, 7, 12 and 24 h post-dose for Compound V and 3 and 12 h for CC-156, from three rats per time point. After the collection at 7 h, a replacement fluid (0.9% sodium chloride injection USP) was injected in the rat. Blood samples were collected into Microvette potassium-EDTA (ethylenediamine tetraacetic acid)-coated tube (Sarstedt, Aktiegesellchaft & Co, Germany) and centrifuged at 3200 rpm for 10 minutes at 4 'C. The plasma was collected and stored at -80 'C until analyzed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis. [00122] Measurement of Plasma Concentrations 1001231 The concentration of the inhibitors (Compounds I and V) was determined by LC/MS/MS analysis using a procedure developed at CHUQ (CHUL) Research Center (Qudbec, Qc, Canada). Briefly, for extraction from serum, 100 piL of serum sample is transferred to individual tubes and 600 iL of ammonium acetate (I mM) is added. A methanolic solution (50 pL) containing a steroidal internal standard is then added to each tube. Samples are transferred on Strata-X SPE colums (Phenomenex, Torrance, CA, USA), which have been conditioned with 2 mL of methanol and 2 mL of water. Each column is washed with 2 mL of methanol:water (10:90, v/v). The inhibitor is then eluted with 5 mL of methanol containing 5 mM ammonium acetate. Methanol is evaporated at 45 'C under inert atmosphere and the residue dissolved in 100 pL of methanol:water (85:15, v/v). For the steroid analysis, the HPLC system uses a 75 x 4.6-mm, 3-pm reversed-phase Luna Phenyl-Hexyl column (Phenomenex, Torrance, CA, USA) at a flow rate of 0.8 mL/min. The inhibitor is detected using an API 3000 mass spectrometer, equipped with TurbolonSpray (Applied Biosystems, Canada). ESI in positive ion mode was used. The area under the curve (AUC) was calculated using the linear trapezoidal rule.

WO 2012/129673 PCT/CA2012/000316 - 46 [001241 In vivo Estrogenicity Assay 1001251 Female ovariectomized (OVX) BALB/c mice weighing approximately 20 g were obtained from Charles-River, Inc. (St-Constant, Qc., Canada). The animals were housed 5 per cage. Groups of 5 mice were treated with El (0.02 pg in 8% EtOH/92% PG) or 17p-HSDl inhibitor at 10, 50 and 250 pg (0.1 mL s.c.) daily for 7 days. Animals were killed 24 h after administration of the last dose of compound and uteri and vagina were removed, excised of fat and weighed. Total body weights of mice were also recorded. [001261 Inhibition of El-Stimulated T-47D Tumor Growth in Nude OVX Mice (xenograft model) [00127] Female OVX BALB/c athymic nude mice weighing approximately 20 g were obtained from Charles-River, Inc. (St-Constant, Qc., Canada). The animals were housed 5 per cage. For the inhibition of T-47D tumor growth, 24 h after a pre-dose of El (0.1 pg) s.c. per mouse, mice were inoculated s.c. with Ix10 7 T-47D cells in 50 pL Matrigel (BD Biosciences, Bedford, MA) into both flanks of each mouse. T-47D tumor growth was stimulated using 0.1 pg of El s.c. per mouse per day for 15 days. From day 16, animals with tumors were randomized in function of tumor volume and separated into three groups. Group I (control mice) was treated s.c. with 100 pL of vehicle alone (8% EtOH/92% PG) per mouse per day. Group 2 (El 0.1 pg) was treated with El (0.1 pg/day, s.c. per mouse) during 32 days. Group 3 (El 0.1 pg + Compound V 250 pg) was treated with El (0.1 pg/day) and Compound V (250 pg/day) per mouse in a combined s.c. injection for 32 days. The mice were weighed at start and volumes of tumors were determined by external caliper twice a week and the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined. Tumor volume based on caliper measurements was calculated by the modified ellipsoidal formula: Tumor volume = 1/2(length x width 2 ) (Jensen MM, Jorgensen JT, Binderup T, Kjaer A. Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and WO 2012/129673 PCT/CA2012/000316 - 47 reproducible than determined by 18F-FDG-microPET or external caliper. BMC Med Imaging 2008;8:16). At the end of the studies the mice were terminally anaesthetized, final body weights and tumor sizes were determined. Uteri and vagina were removed, excised of fat and weighed (Day JM, Foster PA, Tutill HJ, Parsons MF, Newman SP, Chander SK, et al. 17beta-hydroxysteroid dehydrogenase type 1, and not type 12, is a target for endocrine therapy of hormone-dependent breast cancer. Int J Cancer 2008; 122:1931-40; Husen B, Huhtinen K, Poutanen M, Kangas L, Messinger J, Thole H. Evaluation of inhibitors for 17beta-hydroxysteroid dehydrogenase type I in vivo in immunodeficient mice inoculated with MCF-7 cells stably expressing the recombinant human enzyme. Mol Cell Endocrinol 2006;248:109-13; Husen B, Huhtinen K, Saloniemi T, Messinger J, Thole HH, Poutanen M: Human hydroxysteroid (17-beta) dehydrogenase I expression enhances estrogen sensitivity of MCF-7 breast cancer cell xenografts. Endocrinology 2006;147:5333-9; Messinger J, Hirvela L, Husen B, Kangas L, Koskimies P, Pentikainen 0, et al. New inhibitors of 17beta-hydroxysteroid dehydrogenase type 1. Mol Cell Endocrinol 2006;248:192-8). 1001281 Statistical Analysis 1001291 Statistical significance was determined according to the multiple range test of Duncan-Kramer (Kramer C. Extension of multiple range tests to group with unique numbers of replications. Biometrics 1956;12:307-10). P values which were less than 0.05 were considered as statistically significant. 100130] 17p-HSD1 Inhibitory Ativity 100131] The IC 50 values of Compound V and Compound I were determined using breast cancer T-47D cell line (Fig. 3), which exerts strong endogenous expression of 17J-HSDI (Day JM, Foster PA, Tutill HJ, Parsons MF, Newman SP, Chander SK, et al. 17beta-hydroxysteroid dehydrogenase type 1, and not type 12, is a target for endocrine therapy of hormone-dependent breast cancer. Int J Cancer 2008;122:1931-40). Compound V has a good inhibitory effect on 17p-HSD1 WO 2012/129673 PCT/CA2012/000316 -48 with IC 50 value of 68 nM. As a reference, inhibitor compound I inhibited the enzyme with an IC 50 of 27 nM. This IC 50 value is in agreement with the previous value of 44 [M obtained using the same cell line but a different lot of cells and also with a different number of passages (Laplante Y, Cadot C, Fournier MA, Poirier D. Estradiol and estrone C-16 derivatives as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: blocking of ER+ breast cancer cell proliferation induced by estrone. Bioorg Med Chem 2008; 16:1849-60). [001321 Inhibition of El-Stimulated Cell Proliferation [001331 The effectiveness of Compounds V and I to block the proliferative effect induced by El in estrogen-sensitive breast cancer cell line T-47D was investigated. The ability of these 17p-HSDI inhibitors to inhibit the cell growth induced by the transformation of El (0.1 nM) into potent estrogen E2 was investigated. This concentration of El is close to the intracellular concentration in breast cancer cells (Pasqualini JR, Cortes-Prieto J, Chetrite G, Talbi M, Ruiz A. Concentrations of estrone, estradiol and their sulfates, and evaluation of sulfatase and aromatase activities in patients with breast fibroadenoma. Int J Cancer 1997;70:639 43). Compound V was able to inhibit the proliferative effect induced by El in a concentration-dependent manner (FIG. 4A). Compound V reduced the cell growth from 250% to 156 and 125 %, at 2.5 and 5 pM respectively. [001341 The reduction of El-induced cell proliferation obtained when using inhibitor Compound V could also be the result of an antiestrogenic activity of this E2 derivative. Indeed, an antiestrogenic compound will block the proliferative (estrogenic) effect of E2 mediated by its action on the estrogen receptor (ER). As illustrated in FIG. 4B, the enzyme inhibitor Compound V does not reverse the proliferative effect on ER+ cells of E2 (0.1 nM) like the pure antiestrogen EM-139 (Levesque C, Merand Y, Dufour JM, Labrie C, Labrie F. Synthesis and biological activity of new halo-steroidal antiestrogens. J Med Chem 1991;34:1624-30) does. This result indicates that Compound V does not work as an antiestrogenic compound, WO 2012/129673 PCT/CA2012/000316 - 49 but acts instead as an inhibitor of El into E2 transformation catalyzed by 17P-HSDI. 100135] Estrogenic Activity on T-47D (ER*) Cell Line and ERa Binding Affinity 1001361 In order to detect any undesirable estrogenic activity of 17p-HSDI inhibitors, cell proliferative assays were carried out on the T-47D cell line which is known to express the estrogen receptor (ER*) (Keydar I, Chen L, Karby S, Weiss FR, Delarea J, Radu M, et al. Establishment and characterization of a cell line of human breast carcinoma origin. Eur J Cancer 1979;15:659-70). Proliferative activity of compounds V and I was evaluated at 0.5, 1, 2.5 and 5 pM (FIG. 5A). It is clear that Compound V was not estrogenic at any concentration tested, which underlines the importance of the 3-bromoethyl chain to remove the undesired estrogenicity. 1001371 Having assessed the in vitro estrogenic activity of Compound V and I on ER+ cell proliferation, their affinity for ERa (FIG. 5B), the predominant receptor isoform involved in estrogenic effect was investigated. The concentration at which the unlabeled natural ligand (E2) displaces half the specific binding of [ 3

H]

17p-E2 on ERa (ICso) was determined by computer fitting of the data using non-linear regression analysis and the relative binding affinity (RBA) then calculated. The RBA of E2 was established as 100% whereas the RBA for inhibitor Compound I was 1.5%. Although low, this binding affinity for ERa can explain the proliferative (estrogenic) activity we have measured in the T-47D estrogen-sensitive cell line. No binding affinity was detected for Compound V. [00138] Estrogenic Activity of Inhibitors in Mice 100139] To verify that the lack of estrogenicity of Compound V observed in vitro in the T-47D cell proliferation assay translates into the in vivo setting, the estrogenicity of Compound V was investigated using the OVX mouse model by WO 2012/129673 PCT/CA2012/000316 - 50 measuring the weight of the uterus (FIG. 6A) and vagina (FIG. 6B), two estrogen sensitive (ER*) tissues. For the OVX mice control group (OVX-CTR) a low weight of 22 mg was observed for the uterus. However, when administrated s.c. to OVX mice, El (0.02 ptg/mouse/day) is converted into E2 by 170-HSDI and we observed a 2.5 time increase in uterine weight compared to OVX-CTR (22 mg vs 55 mg; P < 0.01). Weights of the uterus from all Compound V dose groups (10, 50 and 250 hg/mouse/day), were not significantly different to those of the OVX-CTR group after seven days of treatment (25, 24 and 23 mg, respectively). Thus, these results confirmed that Compound V is non-estrogenic in vivo. The measurement of vagina weights clearly demonstrated the same tendency for Compound V as previously observed with the uterus. 1001401 Plasma Concentration of Inhibitors [001411 A single subcutaneous injection (2.3 mg/kg) of inhibitors Compounds V and I was given to two different groups of rats in order to determine the inhibitor bioavailability. The mean plasma concentrations of inhibitors Compounds V and I at different times and the corresponding area under the curve (AUC) are presented in FIG. 7. The plasma concentrations at each sampling time were compared to determine the times at which significant differences occurred. At first, it was found that the maximum plasma concentration (Cma) was attained at 3 h following injection for both inhibitors. For Compound V, values of 73.8 and 50.7 ng/mL were found after 7 and 12 h of injection respectively. After 24 h, a plasma concentration of 11.7 ng/mL was measured for Compound V, thus an AUCO- 2 4 h Of 1146 ng*h/mL was obtained for it. [00142] Inhibition of El-Stimulated T-47D Tumor Growth in OVX Nude Mice [00143] After it was established that Compound V was found in plasma after a one-day single s.c. injection, the efficacy of Compound V in vivo was WO 2012/129673 PCT/CA2012/000316 -51 investigated. Female OVX Balb/c nude mice were inoculated with lx107 T-47D (ER*) cells in Matrigel, as in the procedure described by Day et al (Day JM, Foster PA, Tutill HJ, Parsons MF, Newman SP, Chander SK, et al. l7beta-hydroxysteroid dehydrogenase type 1, and not type 12, is a target for endocrine therapy of hormone dependent breast cancer. Int J Cancer 2008; 122:1931-40), except that inoculation was made into both flanks of mouse. The mice received El (0.1 pg/day), which after its transformation to E2 by 17P-HSD1, stimulates tumor growth. Only mice with tumors which were well established after 15 days of treatment with 0.1 ptg El/mouse s.c. were selected to continue the study. We used the dose of 250 pg/mouse of Compound V because this was the highest dose tested in the in vivo estrogenicity assay that proved to be non estrogenic, FIG. 8 shows the effect of Compound V on the growth of tumors stimulated with 0.1 pg El/mouse/day. In the first 18 days of treatment, the tumors were not actively growing and maintained their initial size at the beginning of treatment. From day 19, the volume of tumors in the control (CTR) group began to decrease until they reached approximately the 74% of the initial volume after 28 days and continued at the same level until day 32 (77%). In the El treated group however, tumors grew reaching 136% of their initial size, whereas in the mice treated El Compound V the growth of the tumors was inhibited (74%), decreasing to the level of the CTR group at the end of treatment (P < 0.01 at days 28 and 32, El- Compound V vs El). Clearly, Compound V blocks the formation of E2 in the tumor through the inhibition of 17p-HSD 1 and thus the tumor growth. 1001441 At the end of the study, the body weights of the mice were recorded and the estrogen-sensitive tissues (uterus and vagina) were taken for analysis. There was no effect of either El or Compound V on mouse weight over the 32-day treatment period (FIG. 9), indicating that there is no apparent toxicity of Compound V at 250 pig/day/mouse (s.c.). Although uterine and vagina weights were increased significantly in both of the El-treated groups (P < 0.01, El and El Compound V vs. CTR), treatment with Compound V had no effect on the El stimulated uterine and vaginal weight increase (FIG. 9B and 9C).

WO 2012/129673 PCT/CA2012/000316 - 52 1001451 Synthesis of 3-(2-hydroxyethyl) estra-1(10), 2, 4-trien-17 dioxolane (2) [001461 To a solution of BH 3 -dimethylsulfide (2.0 M in THF, 10.4 mL) in anhydrous THF (70 mL) at -78*C was added dropwise 3-vinyl-estra-1(10),2,4-trien 17-dioxolane (1) (2.25 g, 6.93 mmol) in THF (5 mL) under an argon atmosphere. The resulting solution was stirred at ambient temperature over a period of 16 h. The solution was then cooled to 0 0 C and aqueous NaHCO 3 (1 M, 27.7 mL) was added, immediately followed by the addition of 30% H202 (11.7 mL). The solution was vigorously stirred over a period of 3 h at room temperature and then diluted with EtOAc (50 mL). The resulting solution was poured into water (200 mL) and extracted with EtOAc (5 x 75 mL). The organic layers were combined, washed with brine, dried with MgSO 4 and evaporated under reduced pressure. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 4:6) to provide 1.18 g (50% yield) of compound 2. 1H NMR (400 MHz, Acetone-d 6 ): 0.88 (s, 18-CH 3 ), 1.27-2.38 (unassigned CH and CH 2 ), 2.73 (t, J = 7.1 Hz, CH2CH20H), 2.82 (m, 6-CH 2 ), 3.62 (br t, Oil), 3.71 (mn, CH 2 CHl 2 OH), 3.87 (m, 2 x CH 2 of dioxolane), 6.92 (s, 4-CH), 6.97 (d, J = 8.0 Hz, 2-CH), 7.19 (d, J = 7.9 Hz, 1-CH); "C NMR (100.6 Hz, Acetone d 6 ): 13.9, 22.1, 25.9, 27.0, 28.4, 28.6, 30.7, 33.9, 39.0, 39.1, 44.1, 45.9, 49.3, 63.1, 64.3, 64.9, 118.8, 125.1, 126.2, 129.4, 136.1, 136.5, 137.7. LRMS for C 22

H

31 0 3 [M+H] 343.4 m/z. [001471 Synthesis of 3-[2-(benzyloxy)ethyllestra-1(10),2,4-trien-17-one [001481 To a solution of compound 2 (1.1 g, 3.5 mmol) in anhydrous DMF (50 mL), was added NaH (60% in oil) (168 mg, 4.2 mmol) at 0*C under an argon atmosphere. The solution was stirred over a period of I h at 0 0 C and benzyl bromide (898 mg, 627 pL, 5.3 mmol) was added in one portion. The solution was subsequently allowed to warm to room temperature and was subsequently stirred overnight, poured WO 2012/129673 PCT/CA2012/000316 - 53 into water (300 mL) and extracted with EtOAc (3 x 75 mL), The organic layers were combined, washed with brine, dried with MgSO 4 and evaporated under reduced pressure. The crude compound was then treated with an aqueous solution of HCI (10%) in acetone (1:1) (50 mL) and stirred over a period of 5 h at room temperature. The resulting solution was neutralized using an aqueous NaHCO 3 (10%) solution and extracted with EtOAc (2 x 75 mL). The organic layers were combined, washed with brine, dried with MgSO 4 and evaporated under reduced pressure. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 1:9) to provide 1.04 g (73% yield, 2 steps) of compound 3. 'H NMR (400 MHz, CDC1 3 ): 0.91 (s, 18

CH

3 ), 1.20-2.40 (unassigned CH and CH 2 ), 2.51 (dd, Ji = 8.5 Hz, J 2 = 18.9 Hz, 16$ CH), 2.88 (in, 6-CH 2 and CH 2

CH

2 0), 3.68 (t, J = 7.3 Hz, CH 2 CH2O), 4.54 (s,

OCH

2 Ph), 6.97 (s, 4-CH), 7.02 (d, J = 8.0 Hz, 2-CH), 7.22 (d, J = 7.9 Hz, 1-CH), 7.23-7.38 (in, 5H, OCH 2 Ph); 1 3 C NMR (100.6 Hz, CDCl 3 ): 13.8, 21.6, 25.7, 26.5, 29.4, 31.6, 35.8, 35.9, 38.2, 44.3, 48.0, 50.5, 71.3, 72.9, 125.3, 126.3, 127.5, 127.6, 128.3, 129.6, 136.3 (2x), 136.4 (2x), 137.6, 138.4, 232.5. LRMS for C 27

H

33 0 2 [M+H]* 389.4 m/z. [001491 Synthesis of 3-{(E)-[(16E)-3-[2-(benzyloxy)ethyl]-17-oxoestra 1(10),2,4-trien-16-ylidenelmethyl}benzamide (4) [001501 To a solution of compound 3 (400 mg, 1.03 mmol) in EtOH (25 mL) was added 3-formyl-benzamide (344 mg, 2.05 mmol) and an aqueous KOH (10%) solution (4.5 mL). The resulting reaction mixture was heated at reflux over a period of 30 min. The resulting solution was diluted with water (200 mL), neutralized with aqueous HCI (10%), and extracted with EtOAc (3 x 50 mL). The organic layers were combined, washed with brine, dried with MgSO 4 and evaporated under reduced pressure to provide 400 mg (76 % yield) of compound 4. 1H NMR (400 MHz, CDCl 3 ): 1.00 (s, 18-CH 3 ), 1.25-2.65 (unassigned CH and CH 2 ), 2.89 (t, J = 7.3 Hz,

CH

2

CH

2 O), 2.93 (in, 6-CH 2 ), 3.00 (in, 15-H) 3.69 (t, J = 7.3 Hz, CH 2

CH

2 0), 4.54 (s, OCH2Ph), 5.82 and 6.17 (2 broad s, NH 2 ), 6.99 (s, 4-CH), 7.03 (d, J 8.0 Hz, 2-CH), 7.22-7.38 (in, 5H, 4H of OCH 2 Ph and IH of 1-CH), 7.49 (d, IH, J 3.7 Hz, l'-CH), WO 2012/129673 PCT/CA2012/000316 - 54 7.52 (t, J = 7.7 Hz, 1H of OCH 2 Ph), 7.71 (d, J = 7.8 Hz, 6"-CH), 7.77 (d, J = 7.8 Hz, 4"-CH), 8.04 (s, 2"-CH); "C NMR (100.6 Hz, CDCl 3 ): 14.5, 25.7, 26.9, 29.1, 29.3, 31.7, 35.8, 37.8, 44.3, 47.9, 48.6, 71.2, 72.9, 125.3, 126.4, 126.9, 127.5, 127.6, 127.7, 128.4 (2x), 129.0, 129.2, 129.6, 131.9, 133.5, 133.8, 136.2, 136.3, 136.4, 137.4, 137.5, 138.4, 168.9, 209.4. LRMS for C 35

H

37

NO

3 Na [M+Na]f 542.4 m/z. [001511 Synthesis of 3-11(17p)-17-hydroxy-3-(2-hydroxyethyl)estra 1(10),2,4-trien-16-yllmethyl}benzamide (IV) [001521 To a solution of compound 4 (390 mg, 0.69 mmol) in a mixture of MeOH and DCM (4:1) was added NaBH4 (85 mg, 2.23 mmol). The solution was stirred at room temperature over a period of I h. The resulting solution was concentrated under vacuo, diluted with DCM (30 mL), washed with water, dried with MgSO 4 and evaporated under reduced pressure to provide 375 mg of the crude 17p alcohol. The crude 17p-alcohol was subsequently dissolved in EtOH (100 mL) under an argon atmosphere followed by the addition of Pd on charcoal (10%) (80 mg). The reaction vessel was flushed three times with H 2 and stirred over a period of 36 h, then filtered on celite and evaporated under reduced pressure. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 4:6) to provide 255 mg (84% yield, 2 steps) of compound IV. 'H NMR (400 MHz, CD 3 0D): 0.90 (s, 18-CH 3 ), 1.10-2.55 (unassigned CH and CH 2 ), 2.74 (t, J = 7.2 Hz, CH 2

CH

2 OH), 2.79 (m, 6

CH

2 ), 3.17 (dd, JI = 2.7 Hz, J 2 = 12.5 Hz, IH), 3.71 (t, J = 7.2 Hz, CH 2

CH

2 OH), 3.83 (d, J = 9.4 Hz, 17a-H), 6.89 (s, I H, 4-CH), 6.96 (d, J = 8.0 Hz, 2-CH), 7.19 (d, J = 8.0 Hz, 1-CH), 7.35-7.45 (m, 5"-CH and 6"-CH), 7.70 (d, J = 7.0 Hz, 4"-CH), 7.75 (s, 2"-CH); "C NMR (100.6 Hz, CD 3 0D): 13.3, 27.4, 28.6, 30.5, 33.0, 38.9, 39.0, 39.7, 39.8, 43.3, 45.4, 50.0, 64.4, 83.0, 126.0, 126.2, 127.2, 129.1, 129.2, 129.4, 130.4, 133.5, 134.8, 137.2, 137.5, 139.3, 144.3, 172.0. LRMS for C 28

H

36

NO

3 [M+H] 434.4. 100153] Synthesis of 3-{[(16 ,17 )-3-(2-bromoethyl)-17-hydroxyestra 1(1O),2,4-trien-16-yllmethyllbenzamide

(V)

WO 2012/129673 PCT/CA2012/000316 - 55 [001541 To a solution of compound IV (175 mg, 0.40 mmol) in DCM (15 mL) was added at 0*C triphenylphosphine (200 mg, 0.76 mmol) and carbon tetrabromide (252 mg, 0.76 mmol). The solution was stirred at 0 0 C over a period of 40 min followed by a further addition of triphenylphosphine (100 mg, 0.38 mmol) and carbon tetrabromide (126 mg, 0.38 mmol). The solution was stirred for an addition hour while at 0*C. The resulting mixture was poured into water (150 mL), extracted with DCM (50 mL), dried with MgSO 4 and evaporated under reduced pressure. The crude compound was purified by flash chromatography (DCM/MeOH: 97:3) to provide 168 mg (84% yield) of compound V. 'H NMR (400 MHz, CD 3 0D): 0.91 (s, 18-CH 3 ), 1.10-2.55 (unassigned CH and CH 2 ), 2.82 (in, 6-CH 2 ), 3.06 (t, J = 7.3 Hz,

CH

2

CH

2 Br), 3.17 (dd, Ji = 2.7 Hz, J 2 = 12.5 Hz, 1H), 3.55 (t, J= 7.2 Hz, CH 2

CH

2 Br), 3.84 (d, J = 9.4 Hz, 17a-H), 6.91 (s, 4-CH), 6.97 (d, J = 8.0 Hz, 2-CH), 7.22 (d, J = 8.0 Hz, 1-CH), 7.36-7.44 (m, 5"-CH and 6"-CH), 7.69 (d, J = 7.0 Hz 4"-CH), 7.75 (s, 2"-CH); "C NMR (100.6 Hz, CD 3 0D):13.3, 27.3, 28.5, 30.5, 33.0, 34.0, 38.8, 38.9, 39.6, 40.1, 43.3, 45.4, 45.7, 50.0, 83.0, 126.0, 126.4, 127.0, 129.1, 129.4, 130.1, 133.5, 134.8, 137.4, 137.8, 140.0, 144.3, 175.1. LRMS for C 28

H

3 4

NO

2 [M+H - Br]+ 496.0 and 498.1; HPLC (MeOH/H 2 0: 70:30): 98.5% purity. [001551 Synthesis of 3-{[(16,170)-3-(2-chloroethyl)-17-hydroxyestra 1(10),2,4-trien-16-yllmethyllbenzamide (9) 1001561 To a solution of compound 7 (20 mg, 0.05 mmol) in DCM (1.0 mL) was added chlorodimethyl(phenylthio)-chloride methanaminium (CPMA) (45 mg, 0.19 mmol) at 0 0 C under an argon atmosphere. The solution was then allowed to return to room temperature and stirred for an additional 3 h. The crude compound was directly purified by flash chromatography (DCM/MeOH: 97:3) to provide 12 mg (57%) of compound 9. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.48 (residual CH and CH 2 ), 2.82 (m, 2H, 6-CH 2 ), 2.96 (t, 2H, J = 7.3 Hz, 3-CH 2

CH

2 Cl), 3.19 (in, IH), 3.69 (t, 2H, J = 7.40 Hz, 3-CH 2

CH

2 CI), 3.84 (d, IH, J= 9.4 Hz, 17a-H), 6.92 (s, 1H, 4-CHar), 6.98 (d, 1H, CHar, J = 8.0 Hz), 7.22 (d, IH, CHar, J = 8.0 Hz), 7.36 7.44 (m, 2H, CHar-benzamide), 7.69 (d, lH, CHar-benzamide, J = 7.0 Hz), 7.75 (s, WO 2012/129673 PCT/CA2012/000316 - 56 1H, CHar-benzamide); 1 3 C NMR (MeOD): 13.3, 27.3, 28,6, 30.5, 33.0, 38.8, 39.0, 39.6, 39.8, 43.4, 45.4, 45.7, 46.0, 50.0, 83.0, 126.0, 126.4, 127.1, 129.1, 129.4, 130.3, 133.6, 134.8, 136.7, 137.8, 140.0, 144.4, 172.7. 1001571 Synthesis of 3-{[(16p,17p)-17-hydroxy-3-(2-iodoethyl)estra 1(10),2,4-trien-16-yllmethyl}benzamide (10) 1001581 To a solution of compound 8 (35 mg, 0.07 mmol) in acetone (5 mL) was added sodium iodide (15 mg, 0.1 mmol). The solution was stirred at room temperature under an argon atmosphere over a period of 24 h followed by the addition of another portion of sodium iodide (52 mg, mmol). The solution was the stirred for an additional 24 h. The reaction mixture was subsequently poured into water (100 mL), and extracted three times with EtOAc (3 x 25 mL). The combined organic layer were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 18 mg (47 %) of compound 10. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.03 (residual CH and CH 2 ), 2.81 (m, 2H, 6-CH 2 ), 3.07 (t, 2H, J = 7.7 Hz, 3-CH 2

CH

2 I), 3.19 (in, IH), 3.35 under the MeOD peak (t, 2H, 3-CH 2

CH

2 I), 3.84 (d, 1H, J = 9.4 Hz, 17a-H), 6.92 (s, 1H, 4-CHar), 6.95 (d, IH, CHar, J = 8.0 Hz), 7.21 (d, 1 H, CHar, J = 8.0 Hz), 7.37 7.42 (m, 2H, CHar-benzamide), 7.69 (d, IH, CHar-benzamide, J = 7.0 Hz), 7.76 (s, IH, CHar-benzamide); "C NMR (MeOD): 4.8, 11.9, 25.9, 27.2, 29.1, 31.6, 37.4, 37.6, 38.2, 39.7, 42.0, 44.0, 44.3, 48.6, 81.6, 124.6, 125.1, 125.2, 127.7, 128.0, 128.4, 132.1, 133.4, 136.5, 137.8, 138.6, 143.0, 171.3. [001591 Synthesis of 3-11(163,1 7 p)- 3 -ethenyl-17-hydroxyestra 1(10),2,4-trien-16-yllmethyl}benzamide (11) 100160] To a solution of compound 10 (20 mg) in anhydrous dioxane (1.5 mL) was added TBAF (1.0 M in THF, 37 mg) at room temperature under an argon atmosphere. The solution was then stirred at room temperature over a period of 3h.

WO 2012/129673 PCT/CA2012/000316 - 57 The resulting solution was then poured into water (100 mL) and extracted three times with EtOAc (3 x 25 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 8 mg (53%) of compound 11. 1H NMR (CDCl 3 ):0.88 (s, 3H, 18-CH 3 ), 1.11-2.49 (residual CH and CH 2 ), 2.83 (in, 2H, 6-CH 2 ), 3.18 (in, 1H), 3.87 (d, 1H, J = 9.5 Hz, 17ct-H), 5.18 (d, lH, CH 2 =CH-, J = 10.9 Hz), 5.69 (d, IH, CH 2 =CH-, J = 17.5 Hz), (5.7 and 6.2, 2 br s, 2H, CONH 2 ), 6.66 (dd, I H, CH 2 =CH, JI = Hz, J 2 Hz), 7.11 (s, 1H, CHar), 7.20 (d, I H, CHar, J 8.2 Hz), 7.26 (d, under solvent peak, I H, CHar), 7.38 (in, 2H, CHar), 7.40 (d, IH, J 7.2 Hz, CHar), 7.71 (s, 1H, CHar); 1C NMR (MeOD): 13.3, 27.3, 28.6, 30.5, 33.0, 38.9, 39.0, 39.6, 43.4, 45.4, 45.9, 50.0, 83.0, 112.8, 124.4, 126.0, 126.4, 127.8, 129.1, 129.4, 133.5, 134.8, 136.2, 137.8, 138.2, 141.4, 144.4, 170.0. 1001611 Synthesis of 3-{[(16@,17 )-17-hydroxy-3-(prop-2-en-1 yloxy)estra-1(10),2,4-trien-16-yllmethyllbenzamide (13) [00162] To a solution of compound 12 (150 mg, 0.37 mmol) in acetone (3 mL) was added NaOH (50 mg, 1.25 mmol) and allyl bromide (40 [tL, 0.46 mmol). The resulting solution was then stirred at 60'C over a period of 5h. The reaction mixture was then diluted with EtOAc and washed with a saturated solution of ammonium chloride. The organic layer was washed with brine, dried with MgSO 4 and concentrated to provide 165 mg (99%) of compound 13. Crude compound 13 was found to be sufficiently pure to be used in the next step without further purification. H NMR (Acetone-d 6 ): 0.87 (s, 3H, 18-CH3), 1.07-2.47 (residual CH and CH 2 ), 2.79 (in, 2H, 6-CH 2 ), 3.15 (m, IH), 3.83 (d, IH, J = 10.2 Hz, 17a-H), 4.49 (d, 2H,

OCH

2

CH=CH

2 , J = 5.3 Hz), 5.26 (d, I H, J = 10.4 Hz, CH 2 =CH), 5.39 (d, IH, J = 17.3 Hz, CH 2 =CH), 6.04 (in, IH, CH 2 =CH), 6.12 and 6.33 (2 br s, CONH 2 ), 6.62 (s, IH, CHar), 6.71 (dd, 1H, J, = 2.4 Hz, J 2 = 8.6 Hz), 7.18 (d, 1H, CHar, J = 8.6 Hz), 7.33 (in, 2H, CHar), 7.60 (d, IH, CHar, J = 7.3 Hz), 7.71 (s, 1 H, CHar).

WO 2012/129673 PCT/CA2012/000316 - 58 [001631 Synthesis of 3-{[(16D,17B)-17-hydroxy-3-(2 hydroxyethoxy)estra-1(10),2,4-trien-16-yllmethylIbenzamide (14) 100164] Sodium periodate (108 mg, 0.50 mmol) was added to water (0.5 mL) and stirred at 0 0 C over a period of 5 min, followed by subsequent addition of RuCl 3

-H

2 0 (4 mg, 0.02 mmol), EtOAc (1 mL) and acetonitrile (1 mL). Compound 13 (150 mg, 0.33 mmol) was then added to the solution and the reaction mixture stirred for about 2 minutes. The reaction mixture was then quenched by the addition of a saturated aqueous solution of Na 2

S

2

O

3 (2 mL). The phases were separated and the aqueous layer extracted with EtOAc (3 x 3 mL). The combined organic layers were dried with Na 2

SO

4 and concentrated. The residue was then dissolved in a mixture of THF (1 mL) and water (1 mL), followed by the addition of NaBH4 (13 mg, 0.34 mmol). The reaction mixture was stirred at room temperature over a period of 20 min followed by the subsequent addition of water (10 mL). The reaction mixture was then extracted with DCM (3 x 15 mL), the combined organic layers washed with a saturated bicarbonate solution, dried with Na 2

SO

4 , and concentrated. The residue was dissolved in THF (1 mL) and water (I mL) at 0 0 C followed by the addition of sodium periodate (144 mg, 0.67 mmol) in small portions. The solution was subsequently stirred over a period of 20 min at room temperature. Ethylene glycol (50 PL) was then added and the reaction mixture diluted with water (3 mL). The reaction mixture was extracted with EtOAc (3 x 5 mL) and the combined organic layers dried with Na 2

SO

4 and concentrated. The residue was redissolved in a mixture of THF (1 mL) and water (1 mL) followed by the addition NaBH 4 (13 mg, 0.34 mmol). The resulting reaction mixture was stirred at room temperature over a period of lh followed by the addition of water (I mL). The reaction mixture was extracted with DCM (3 x 5 mL) and the combined organic layers washed with a saturated bicarbonate solution, dried with Na 2

SO

4 , and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 9:1) to provide 25 mg (15%) of compound 14. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH3), 0.96-2.48 (residual CH and CH 2 ), 2.79 (in, 2H, 6-CH 2 ), 3.18 (in, IH), 3.84 (in, 3H, OCH 2

CH

2 OH and 17a-H), 3.99 (in, 2H,

OCH

2

CH

2 OH), 6.69 (d, I H, 4-CHar, J = 2.8 Hz), 6.72 (d, 1 H, CHar, J = 2.7 Hz), 7.17 WO 2012/129673 PCT/CA2012/000316 - 59 (d, 1H, CHar, J = 8.0 Hz), 7.38-7.42 (m, 2H, CHar-benzamide), 7.69 (d, 1H, CHar benzamide, J = 7.4 Hz), 7.76 (s, I H, CHar-benzamide). 1001651 Synthesis of 3-{[(16 ,17 )-3-(2-bromoethoxy)-17 hydroxyestra-1(10),2,4-trien-16-yllmethylbenzamide (15) [001661 To a solution of compound 14 (20 mg, 0.46 mmol) in a mixture of anhydrous DCM (2 mL) and anhydrous THF (1 mL) was added at 0 0 C triphenylphosphine (23 mg, 0.87 mmol) and carbon tetrabromide (29 mg, 0.87 mmol). The reaction mixture was stirred at 0 0 C over a period of 40 min followed by a further addition of triphenylphosphine (20 mg, 0.46 mmol) and carbon tetrabromide (23 mg, 0.87 mmol). The reaction mixture was stirred at 0 0 C for an additional 40 min followed by the further addition of triphenylphosphine (20 mg, 0.46 mmol) and carbon tetrabromide (23 mg, 0.87 mmol). The reaction mixture was then stirred for an additional hour at 0 0 C. The reaction mixture was subsequently poured into water (100 mL) and extracted with DCM (2 x 25 mL). The combined organic layers were dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/ ether/MeOH: 75:20:5) to provide 12 mg (52 %) of compound 15. 1H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.13-2.48 (residual CH and CH 2 ), 2.78 (m, 2H, 6-CH 2 ), 3.17 (m, 1H), 3.67 (t, 2H, J = 7.2 Hz, 3-OCH 2

CH

2 Br, J = Hz), 3.84 (d, I H, J = 9.4 Hz, 17a-H), 4.25 (t, 2H, 3-OCH 2

CH

2 Br, J = 5.7 Hz), 6.62 (s, 1 H, 4 CHar), 6.69 (d, 1 H, CHar, J = 8.7 Hz), 7.19 (d, 1H, CHar, J = 8.6 Hz), 7.38-7.42 (m, 2H, CHar-benzamide), 7.69 (d, I H, CHar-benzamide, J = 7.4 Hz), 7.76 (s, 1H, CHar benzamide); 1C NMR (CDCl 3 ): 13.3, 27.5, 28.6, 30.7 (2x), 33.0, 38.8, 39.0, 39.8, 43.3, 45.4 (2x), 49.9, 69.2, 83.0, 113.3, 115.6, 126.0, 127.4, 129.1, 129.4, 133.6, 134.5, 134.8, 139.1, 144.4, 157.5, 172.4. [00167] Synthesis of (8R, 9S, 13S, 14S)-13-methy-3-[(E)-2 phenylethenyll-6,7,8,9,11,12,13,14,15,16 decahydrospirofcyclopentalalphenanthrene-17,2'-[1,3ldioxolanel (16) WO 2012/129673 PCT/CA2012/000316 - 60 [001681 To a solution of compound 1 (350 mg, 1.07 mmol) in DCM (75 mL) under an argon atmosphere was added styrene (257 ptL, 233 mg, 2.24 mmol). The solution was purged by argon bubbling over a period of 5 min followed by the addition of Grubb (1I) catalyst (48 mg, 0.056 mmol). The reaction mixture was subsequently refluxed over a period of 24 h under an argon atmosphere. The reaction mixture was then poured into water, extracted twice with DCM (2 x 50 mL), filtered using a phase separator device (Biotage) and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 95:5) to provide 50 mg (11%) of compound 16. 1H NMR (CDCl 3 ): 0.89 (s, 3H, 18-CH 3 ), 1.34-2.39 (residual CH and

CH

2 ), 2.90 (in, 2H, 6-CH 2 ), 3.89 (in, 4H, 2 x CH 2 of dioxolane), 7.19 (s, 2H, ), 7.23 7.38 (in, 6H, CHar and CH=CH), 7.58 (d, 2H, CHar, J = 8.2 Hz). [00169] Synthesis of 3-{{E)-1(16E)-17-oxo-3-I(E)-2 phenylethenyllestra-1(10),2,4-trien-16-ylidenelmethyllbenzamide (17) [00170] To a solution of compound 16 (42 mg, 0.105 mmol) in methanol (3 mL) was added an aqueous solution of HCI 10% (1 mL). The reaction mixture was then stirred at room temperature over a period of 2 h. The reaction mixture was then poured into a sodium bicarbonate solution (50 mL) and extracted twice with EtOAc (2 x 20 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated to provide 40 mg of deprotected ketone product. 'H NMR (CDCl 3 ): 0.92 (s, 3H, 18-CH 3 ), 1.25-2.53 (residual CH and CH 2 ), 2.95 (m, 2H, 6-CH 2 ), 7.08 (s, 2H), 7.23-7.38 (in, 6H, CHar + CH=CH), 7.51 (d, 2H, J = 8.4 Hz). The crude ketone compound (38 mg) was then dissolved in ethanol (5 mL) followed by the addition of 3-formyl-benzamide (34 mg, 0.227 mmol) and an aqueous KOH solution (10%, 0.6 mL). The reaction mixture was then heated at reflux over a period of 60 min. The resulting reaction mixture was then diluted with water (50 mL), neutralized with an aqueous HCl solution (10%), and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated to provide 40 mg (72 %) of compound 17. 'H NMR (CDCl 3 ): 1.03 (s, 3H, CH 3 -18), 1.25-2.67 (residual CH and CH 2 ), 2.99 (in, 2H, 6-CH 2 ), 5.7 and 6.1 (br s, 2H, WO 2012/129673 PCT/CA2012/000316 -61

CONH

2 ), 7.08 (s, I H), 7.23-7.52 (m, 8H, CHar and PhCH=CHPh), 7.71 (d, I H, CHar, J = Hz), 7.77 (d, 1 H, CHar, J = Hz), 8.04 (s, IH, CHar). 100171] Synthesis of 3-{(E)-[(16E,17p)-17-hydroxy-3-[(E)-2 phenylethenyllestra-1(10),2,4-trien-16-ylidenelmethyllbenzamide (18) 1001721 To a solution of compound 17 (40 mg, 0.082 mmol) in MeOH (3 mL) was added NaBH 4 (10 mg, 0.26 mmol). The reaction mixture was then stirred at room temperature over a period of I h and subsequently poured into water and extracted twice with EtOAc (20 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 95:5) to provide 24 mg (60%) of compound 18. 'H NMR (CDCl 3 ): 0.76 (s, 3H, 18-CH 3 ), 0.79-2.46 (residual CH and CH 2 ), 2.80 (m, 2H, 6-CH 2 ), 4.18 (d, IH, 17a-H, J = 9.0 Hz), 5.7 and 6.1 (br s, 2H, CON1 2 ), 6.60 (s, IH, C=CH-benzamide), 7.08 (s, 2H, CHar), 7.23-7.62 (m, 8H, CHar and PhCH=CHPh), 7.71 (d, I H, CHar, J = 7.8 Hz), 7.77 (d, I H, CHar, J 7.8 Hz), 8.04 (s, IH). [00173] Synthesis of 3-{[(160,17 )-17-hydroxy-3-(2-phenylethyl)estra 1(10),2,4-trien-16-yllmethyllbenzamide (19) [001741 To a solution of compound 18 (20 mg, 0.041 mmol) in a mixture of EtOH/DCM (1:1) (3 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (5 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 24 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 7:3) to provide 6 mg (32%) of compound 19. 1 H NMR (CDCl 3 ): 0.88 (s, 3H, 18-CH 3 ), 0.91-2.50 (residual CH and

CH

2 ), 2.77-2.93 (m, 6H, 6-CH 2 and 2 x CH 2 Ph), 3.17 (m, IH), 3.87 (m, IH), 5.7 and 6.1 (br s, 2H, CONH 2 ), 6.94 (s, 1H), 7.19 (dd, 1H, JI = 7.9 Hz, J 2 = 1.3 Hz), 7.22-7.53 (m, 7H, CHar), 7.60 (d, 1H, J = 5.9 Hz), 7.72 (s, I H); "C NMR (MeOD): 13.3, 27.4, WO 2012/129673 PCT/CA2012/000316 - 62 28.7, 30.5, 33.0, 38.7, 38.8, 39.0, 39.2, 39.7, 43.4, 45.4, 45.7, 50.0, 83.0, 126.0, 126.1, 126.8 (2x), 129.1, 129.2 (2x), 129.4, 129.5 (2x), 129.9, 133.5, 134.8, 137.4, 139.0, 140.1, 143.3, 144.4, 167.6. [00175] Synthesis of (8R, 9S, 13S, 14S)-3-[(1E)-3-(benzyloxy)prop-1 en-1-yll-13-methyl-6,7,8,9,11,12,13,14,15,16 decahydrospirocyclopentalalphenanthrene-17,2'-[1,3jdioxolanel (20) [001761 To a solution of compound 1 (1.5 g, 4.82 mmol) in DCM (400 mL) under an argon atmosphere was added [(prop-2-en-1-yloxy)methyl] benzenebenzyl prop-2-en-1-yl ether (1.3 g, 9.55 mmol). The reaction mixture was subsequently stirred over a period of 5 min followed by the addition of Grubb (II) catalyst (204 mg, 0.24 mmol). The resulting solution was then stirred at 60'C while under an argon atmosphere over a period of 48 h. The reaction mixture was then concentrated and purified by flash chromatography using EtOAc/Hexanes (5:95) as eluant to provide 130 mg (6%) of compound 20. 'H NMR (CDCl 3 ): 0.91 (s, 3H, 18

CH

3 ), 1.25-2.42 (residual CH and CH 2 ), 2.78 (in, 2H, 6-CH 2 ), 3.74-4.06 (in, 4H, 2 x CH2 of dioxolane), 4.18 (d, 2H, J = 6.1 Hz, CH 2

OCH

2 Ph), 4.56 (s, 2H, CH 2 0CH 2 Ph), 6.24-6.31 (m, 1H, CH=CHCH 2 O), 6.57 (d, 1H, J = 16.2 Hz, CH=CHCH 2 0), 7.11 (s, 1H, CH ar), 7.18 (d, 1 H, J = 8.2 Hz, CHar), 7.25-7.39 (m, 6H, CHar). [00177] Synthesis of 3- I(1E)-3-(benzyloxy)prop- 1-en-1-yll estra 1(10),2,4-trien-17-one (21) [001781 To a solution of compound 20 (120 mg, 0.28 mmol) in acetone (3 mL) was added an aqueous solution of HC (10%, 3 mL). The reaction mixture was then stirred at room temperature over a period of 6 h. The reaction mixture was then diluted with water (60 mL), neutralized with bicarbonate solution and extracted three times with EtOAc (3 x 20 mL). The combined organic layers washed with brine, dried with MgSO 4 , and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 5:95) to provide 80 mg (69%) of compound 21. 'H WO 2012/129673 PCT/CA2012/000316 - 63 NMR (CDC1 3 ): 0.91 (s, 3H, 18-CH 3 ), 1.22-2.32 (residual CH and CH 2 ), 2.52 (dd, IH, J,= 19.1 Hz, J 2 = 8.9 Hz, 16p-CH), 2.92 (in, 2H, CH 2 -6), 4.19 (d, 2H, J = 6.1 Hz,

OCH

2 CH=CH), 4.57 (s, 2H, OCH 2 Ph), 6.26-6.33 (in, 1H, CH=CHCH 2 0), 6.58 (d, 1H, J = 16.0 Hz, CH=CHCH 2 0), 7.14 (s, IH, CH ar), 7.19 (d, I H, J = 8.2 Hz, CHar), 7.25-7.39 (in, 6H, CHar). [00179] Synthesis of 3-((E)-U(16E)-3-(1E)-3-(benzyioxy)prop-1-en-1 yll-17-oxoestra-1(10),2,4-trien-16-ylidenelmethyl}benzamide (22) 1001801 To a solution of compound 21 (80 mg) in EtOH (10 mL) was added 3-formyl-benzamide (62 mg) and an aqueous KOH solution (10%, 1.7 mL). The reaction mixture was then heated at reflux over a period of 30 min. The resulting reaction mixture was then diluted with water (100 mL), neutralisazed with an aqueous HCI solution (10%), and extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 1:1) to provide 65 mg (61%) of compound 22. 'H NMR (MeOD): 1.05 (s, 3H, 18-CH 3 ), 0.89-3.24 (residual CH and CH 2 ), 4.20 (d, 2H, J = 6.1 Hz, OCH 2 CH=CH), 4.58 (s, 2H,

OCH

2 Ph), 6.26-6.33 (in, lH, CH=CHCH 2 0), 6.58 (d, 1H, J = 16.0 Hz,

CH=CHCH

2 0), 7.16 (s, 1H, CH ar), 7.21 (d, IH, J = 8.1 Hz, CHar), 7.25-7.39 (in, 6H, CHar) 7.49 (s, 1H, CHar-benzamide), 7.59 (t, 2H, OCH 2 Ph, J = 7.7 Hz), 7.81 (d, IH, CHar of benzamide, J = 7.7 Hz), 7.91 (d, IH, CHar-benzamide, J = 8.2 Hz), 8.14 (s, IH, CHar-benzamide). [001811 Synthesis of 3-{(E)-[(1 6 E,170)-3-1(1E)-3-(benzyloxy)prop-1-en 1-yll-17-hydroxyestra-1(10),2,4-trien-16-ylidenelmethyl)benzamide (23) [001821 To a solution of compound 22 (55 mg, 0.11 mmol) in a mixture of MeOH and DCM (4:1) was added NaBH 4 (12 mg, 0.32 mmol). The reaction mixture was stirred at room temperature over a period of 2 h and concentrated. The residue WO 2012/129673 PCT/CA2012/000316 - 64 was diluted with DCM (15 mL) and washed with water. The organic layer was then dried with MgSO 4 and concentrated to provide 50 mg (91%) of compound 23 (without further purification). 1 H NMR (MeOD): 0.77 (s, 3H, 18-CH 3 ), 0.80-2.84 (residual CH and CH 2 ), 2.89 (in, 2H, CH 2 -6), 4.14 (br s, 1H, 17a-H), 4.19 (d, 2H, J= 6.2 Hz, OCH 2 CH=CH), 4.57 (s, 2H, OCH 2 Ph), 6.27-6.34 (in, 1H, CH=CHCH 2 O), 6.56 (in, 2H, CH=CHCH 2 0 and C=CHPhCONH 2 ), 7.13 (s, 1H, CH ar), 7.20 (d, IH, J = 8.1 Hz, CHar), 7.27-7.42 (in, 6H, CHar) 7.46 (t, IH, J = 7.7 Hz, CHar-benzamide), 7.60 (d, 1H, CHar of benzamide, J = 7.7 Hz), 7.70 (d, IH, CHar-benzamide, J = 8.2 Hz), 7.94 (s, I H, CHar-benzamide). [001831 Synthesis of 3-11(16B,17M)-17-hydroxy-3-(3 hydroxypropyl)estra-1(10),2,4-trien-16-yllmethyllbenzamide (24) [00184] To a solution of compound 23 (45 mg, 0.086 mmol) in a mixture of EtOH/DCM (4:1) (5 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (20 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 36 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography (EtOAc) to provide 38 ing (99%) of compound 24. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.22-2.48 (residual CH and CH 2 ), 2.59 (t, 2H, J = 7.4 Hz, CH 2

CH

2 OH), 2.78 (in, 2H, 6-CH 2 ), 3.16 (m, IH), 3.56 (t, 2H, J = 6.6 Hz, CH 2

CH

2 OH), 3.84 (d, IH, J = 9.4 Hz, 17a-H), 6.87 (s, 1H, 4-CHar), 6.93 (d, 1H, CHar, J = 8.0 Hz), 7.18 (d, 1H, CHar, J = 8.0 Hz), 7.38-7.44 (in, 2H, CHar benzamide), 7.41 (d, IH, CHar-benzamide, J = 7.0 Hz), 7.76 (s, IH, CHar benzamide); 1 3 C NMR (MeOD): 13.3, 27.4, 28.7, 30.5, 32.6, 33.0, 35.5, 38.9, 39.0, 39.8, 43.4, 45.4, 45.7, 50.0, 62.3, 83.0, 126.0, 126.2, 126.7, 129.1, 129.4, 129.9, 133.5, 134.8, 137.5, 138.9, 140.3, 144.4, 172.7. [001851 Synthesis of 3-t (160,170)-3-(3-bromopropyl)-17 hydroxyestra-1(10),2,4-trien-16-yllmethyllbenzamide (25) WO 2012/129673 PCT/CA2012/000316 - 65 [00186] To a solution of compound 24 (28 mg, 0.063 mmol) in DCM (3 mL) at 0 0 C was added triphenylphosphine (33 mg, 0.13 mmol) and carbon tetrabromide (42 mg, 0.13 mmol). The reaction mixture was then stirred at 0 0 C over a period of 40 min followed by the addition of a second portion of triphenylphosphine (13 mg, 0.05 mmol) and carbon tetrabromide (17 mg, 0.05 mmol). The reaction mixture was then stirred for an additional hour while at 0 0 C. The reaction mixture was subsequently poured into water (50 mL) and extracted with DCM (2 x 25 mL). The combined organic layers were then dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 97:3) to provide 8 mg (25%) of compound 25. 'H NMR (CDCl 3 ): 0.88 (s, 3H, 18-CH 3 ), 1.11-2.55 (residual CH and CH 2 ), 2.70 (t, 2H, J = 7.2 Hz, CH 2

CH

2 Br), 2.78 (m, 2H, 6-CH 2 ), 3.16 (m, 1H), 3.41 (t, 2H, J = 6.6 Hz, CH 2

CH

2 Br), 3.88 (m, 1H, 170c-H), 5.6 and 6.2 (2 br s, CONH 2 ), 6.91 (s, IH, 4-CHar), 6.97 (d, 1H, CHar, J = 8.0 Hz), 7.22 (d, 1H, CHar, J = 8.0 Hz), 7.35-7.42 (m, 2H, CHar-benzamide), 7.40 (d, 1H, CHar benzamide, J = 7.0 Hz), 7.72 (s, 1H, CHar-benzamide); 13 C NMR (MeOD): 13.3, 27.4, 28.6, 30.5, 33.0, 33.7, 34.4, 35.7, 38.8, 39.0, 39.7, 43.4, 45.4, 45.7, 50.0, 83.0, 126.0, 126.4, 126.8, 129.1, 129.4, 130.0, 133.8, 134.8, 137.7, 139.0, 139.3, 144.4, 172.7. 1001871 Synthesis of 3-ethenylestra-1(10),2,4-trien-17-one (26) [00188] To a solution of compound 1 (180 mg, 0. mmol) in acetone (18 mL) was added an aqueous solution of HCI (10%, 2 mL). The reaction mixture was then stirred at room temperature over a period of 2 h. The solution was then poured into a sodium bicarbonate solution (50 mL) and extracted twice with EtOAc (2 x 20 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated to provide 140 mg of deprotected ketone product. 'H NMR (Acetone d 6 ): 0.90 (s, 3H, 18-CH 3 ), 1.38-2.48 (residual CH and CH 2 ), 2.86 (in, 2H, 6-CH 2 ), 5.16 (d, I H, PhCH=CH 2 , J = 10.9 Hz), 5.74 (d, I H, PhCH=CH 2 , J = Hz), 6.69 (m, I H, PhCH=CH 2 ), 7.16 (s, IH, CHar), 7.26 (in, 2H, CHar).

WO 2012/129673 PCT/CA2012/000316 - 66 [001891 Synthesis of 3-{(E)-[(16E,17p)-3-ethenyl-17-hydroxyestra 1(10),2,4-trien-16-ylidenelmethyl!benzamide (27) [001901 To a solution of compound 26 (115 mg, 0.40 mmol) in EtOH (10 mL) was added 3-formyl-benzamide (125 mg, 0.84 mmol) and an aqueous KOH solution (10%, 1.7 mL). The reaction mixture was then heated at reflux over a period of 40 min. The resulting reaction mixture was then diluted with water (100 mL), neutralisazed with an aqueous HC solution (10%), and extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 7:3) to provide 80 mg (47%) of compound 27. 'H NMR (CDCl 3 ): 0.90 (s, 3H, 18-CH 3 ), 1.28-2.65 (residual CH and CH 2 ), 2.98 (m, 3H, 6-CH 2 and 15 CH), 5.20 (d, 1H, PhCH=CH 2 , J = 10.9 Hz), 5.72 (d, IH, PhCH=CH 2 , J = 17.5 Hz), 5.9 and 6.2 (2 br s, CONH 2 ), 6.67 (in, IH, PhCH=CH 2 ), 7.16 (s, CHar), 7.24 (in, 2H, CHar), 7.49 (s, IH, CHar), 7.51 (tapp, IH, CHar, J = 7.7 Hz), 7.71 (d, IH, CHar, J = 7.8 Hz), 7.77 (d, I H, CHar, J = 7.8 Hz), 7.79 (s, I H). [00191] Synthesis of 3-f{(E)-1(16E,17p)-3-ethenyl-17-hydroxyestra 1(10),2,4-trien-16-ylidenelmethyl!benzamide (28) [00192] To a solution of compound 27 (80 mg, 0.19 mmol) in a mixture of MeOH and DCM (9:1) was added NaBH4 (22 mg, 0.58 mmol). The reaction mixture was stirred at room temperature over a period of 2 h and concentrated. The residue was diluted with DCM (25 mL) and washed with water. The organic layer was then dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 74 mg (92%) of compound 27. 1 H NMR (MeOD): 0.74 (s, 3H, 18-CH 3 ), 1.26-2.78 (residual CH and CH 2 ), 2.88 (in, 2H, 6-CH 2 ), 4.17 (br d, IH, 17a-H, J = 8.6 Hz), 5.17 (d, IH, PhCH=CH 2 , J = 11.5 Hz), 5.71 (d, 1H, PhCH=CH2, J = 17.5 Hz), 5.7 and 6.2 (2 br s, CONH 2 ), 6.60 (s, IH, 16 CH=Ph), 6.67 (m, IH, PhCH=CH 2 ), 7.15 (s, CHar), 7.21 (d, 2H, CHar, J = 8.1 Hz), WO 2012/129673 PCT/CA2012/000316 - 67 7.28 (d (under solvent peak), 1H, CHar, J = 10.7 Hz), 7.43 (t, 1 H, CHar, J = 7.7 Hz), 7.56 (d, I H, CHar, J = 7.8 Hz), 7.61 (d, I H, CHar, J= 7.8 Hz), 7.87 (s, IH). [001931 Synthesis of 3-{1(16 ,17p)-3-ethyl-17-hydroxyestra-1(10),2,4 trien-16-yllmethyllbenzamide (29) [001941 To a solution of compound 28 (74 mg, 0.18 mmol) in EtOH (5 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (15 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 48 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography (EtOAc) to provide 40 mg (54%) of compound 29. 1H NMR (CDCl 3 ): 0.87 (s, 3H, 18-CH 3 ), 1.10-2.60 (residual CH and CH 2 ), 1.22 (t, 3H,

CH

3

CH

2 Ph), 2.59 (q, 2H, J = 7.4 Hz, CH 3

CH

2 Ph), 2.82 (in, 2H, 6-CH 2 ), 3.16 (in, I H), 3.56 (t, 2H, J = 6.6 Hz, CH 2

CH

2 OH), 3.88 (in, 1H, 17a-H), 5.7 and 6.2 (2 br s,

CONH

2 ), 6.92 (s, 1H, 4-CHar), 6.99 (d, I H, CHar, J = 8.0 Hz), 7.22 (d, I H, CHar, J = 8.0 Hz), 7.37 (in, 2H, CHar-benzamide), 7.60 (d, 1H, CHar-benzamide, J = 7.0 Hz), 7.72 (s, IH, CHar-benzamide); "C NMR (MeOD): 13.3, 16.3, 27.4, 28.7, 29.4, 30.5, 33.0, 38.9, 39.0, 39.7, 43.3, 45.4, 45.6, 50.0, 83.0, 126.0, 126.1, 126.2, 129.1, 129.2, 129.4, 133.5, 134.8, 137.4, 138.6, 142.4, 144.3, 172.7. [00195] Synthesis of [(16$)-16-(3-carbamoylbenzyl)-17-oxoestra 1(10),2,4-trien-3-yllacetic acid (30) [001961 Dess Martin periodane (67 mg, 0.16 mmol) was added in one portion to a solution of alcohol 11 (50 mg, 0.12 mmol) in DCM (4 mL) at room temperature. The reaction mixture was stirred at room temperature over a period of lh and subsequently treated with a saturated aqueous solution of NaHSO 3 (0.25 mL) followed by treatment with NaHCO 3 (5 mL). The aqueous layer was then extracted with EtOAc (2 x 5 mL) and the combined organic layers dried with MgSO 4 , filtered WO 2012/129673 PCT/CA2012/000316 - 68 and concentrated. The crude product (49 mg) was subsequently taken up in t-BuOH (2.2 mL) and water (0.2 mL) followed by the addition of 2-methyl-2-butene (64 PL, 0.76 mmol), NaClO 2 (13 mg, 0.14 mmol) and KH 2

PO

4 (19 mg, 0.14 mmol). The suspension was subsequently stirred over a period of 12 h at room temperature. The organic phase was then concentrated and the aqueous phase acidified with an aqueous solution of HCI (IN, I mL). The aqueous phase was then extracted with EtOAc (3 x 10 mL). The combined organic layers were then washed with brine, dried over MgSO 4 and concentrated. The crude product was purified by trituration from MeOH to provide 30 mg (59%) of compound 30. 1H NMR (Acetone-d6): 0.68 (s, 3H, 18

CH

3 ), 1.33-2.73 (residual CH and CH 2 ), 2.78 (m, 2H, 6-CH 2 ), 3.09 (m, 1H), 3.46 (s, 2H, CH 2 COOH), 6.92 (s, IH, CHar), 6.98 (d, IH, CHar, J = 8.2 Hz), 7.20 (d, IH, CHar, J = 8.0 Hz), 7.36 (m, 2H, CHar), 7.71 (m, 2H, CHar), 12.1 (br, s, COOH). [00197] Synthesis of [(160,17$)-16-(3-carbamoylbenzvl)-17 hydroxyestra-1 (10),2,4-trien-3-yll acetic acid (31) 1001981 To a solution of compound 30 (30 mg, 0.067 mmol) in MeOH (5 mL) was added NaBI-4 (7 mg, 0.18 mmol). The reaction mixture was then stirred at room temperature over a period of 2 h followed by the addition of two further portions (7 mg, 0.18 mg) of NaBH 4 sequentially added over a period of 2 h. The reaction mixture was then concentrated, diluted with DCM (25 mL) and washed with water. The organic layer was dried with MgSO 4 and concentrated. The crude product was purified by flash chromatography (DCM/MeOH: 9:1) to provide 15 mg (50%) of compound 31. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.13-2.49 (residual CH and

CH

2 ), 2.81 (m, 2H, 6-CH 2 ), 3.17 (m, I H), 3.52 (s, 2H, CH 2 COOH), 3.84 (d, I H, 17a H, J = 9.4 Hz), 6.96 (s, IH, CHar), 7.01 (d, IH, CHar, J = 8.0 Hz), 7.23 (d, I H, CHar, J = 8.0 Hz), 7.40 (m, 2H, CHar), 7.69 (d, 2H, CHar, J = 6.0 Hz), 7.75 (s, I H, CHar); "C NMR (MeOD): 13.3, 27.3, 28.6, 30.5, 33.0, 38.8, 39.0, 39.6, 43.4, 45.4, 45.7, 50.0, 83.0, 126.0, 126.4, 127.6, 129.1, 129.4, 130.8, 133.3, 133.6, 134.8, 137.8, 140.1, 144.4, 172.7.

WO 2012/129673 PCT/CA2012/000316 - 69 [00199] Synthesis of 3-1(16 ,17p)-17-hydroxy-3-[2-(methylamino)-2 oxoethyllestra-1(10),2,4-trien-16-vilmethyl} benzamide (32) 1002001 To a solution of compound 30 (37 mg, 0.08 mmol) in anhydrous DMF (3 mL), under an argon atmosphere, was added BOP (40 mg, 0.09 mmol), methyl amine (115 ptL, 0.03 mmol; 2.0 M in THF) and DIPEA (18 pL, 0.11 mmol). The reaction mixture was stirred at room temperature over a period of 3 h, subsequently poured into water and extracted twice with EtOAc (2 x 10 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated to provide 41 mg of crude product. The crude product was taken up into a mixture of methanol/DCM (9:1) followed by the addition of NaBH 4 (15 mg, 0.40 mmol). The reaction mixture was then stirred at room temperature over a period of 30 min and poured into water. The reaction mixture was extracted twice with EtOAc (2 x 10 mL) and the combined organic layers washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 6 mg (15%) of compound 32. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.49 (residual CH and CH 2 ), 2.71 (s, 3H, CH 2

CONHCH

3 ), 2.80 (in, 2H, 6-CH 2 ), 3.16 (in, I H), 3.41 (s, 2H, CH 2

CONHCH

3 ), 3.84 (d, I H, 17a-H, J = 9.4 Hz), 6.96 (s, 1H, CHar), 7.01 (d, IH, CHar, J = 8.0 Hz), 7.23 (d, I H, CHar, J = 8.0 Hz), 7.42 (in, 2H, CHar), 7.69 (d, 2H, CHar, J = 6.0 Hz), 7.76 (s, 1H, CHar); 13C NMR (MeOD): 13.3, 26.5, 27.3, 28.6, 30.5, 33.0, 38.9, 39.0, 39.7, 43.4 (2x), 45.4, 45.7, 50.0, 83.0, 125.4, 126.0, 126.5, 127.3, 129.1, 129.4, 130.5, 133.5, 134.8, 138.0, 140.2, 144.4, 175.1. [002011 Synthesis of (1 6 E)-16-(3-carbamoylbenzylidene)-17-oxoestra 1,3,5(10)-triene-3-carboxylic acid (34) 100202] To a solution of compound 33 (250 mg, 0.84 mmol) in EtOH (10 mL) was added 3-formyl-benzamide (250 mg, 1.67 mmol) and an aqueous KOH solution (10%, 1.7 mL). The reaction mixture was then heated at reflux over a period of 30 min. The resulting reaction mixture was then diluted with water (200 mL), WO 2012/129673 PCT/CA2012/000316 - 70 neutralized with an aqueous HCI solution (10%), and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 143 mg (40 %) of compound 34. 1H NMR (MeOD): 1.06 (s, 3H, 18-CH 3 ), 1.31-2.77 (residual CH and CH 2 ), 3.40 (m, 2H, 6-CH 2 ), 7.42 (d, I H, CHar, J = Hz), 7.50 (s, 1H, CH=CH), 7.59 (t, I H, CHar, J = 7.8 Hz), 7.77 (d, IH, CHar, J = 10.2 Hz), 7.83 (d, 1H, CHar, J = 7.7 Hz), 7.91 (d, IH, CHar, J = 7.8 Hz), 8.14 (s, I H, CHar). 1002031 Synthesis of (16E,170)-16-(3-carbamoylbenzylidene)-17 hydroxyestra-1,3,5(10)-triene-3-carboxylic acid (35) 1002041 To a solution of compound 34 (140 mg, 0.33 mmol) in a mixture of MeOH and DCM (1:1) was added NaBH 4 (21 mg, 0.55 mmol). The reaction mixture was stirred at room temperature over a period of 2h and concentrated. The residue was diluted with DCM (25 mL) and washed with water. The organic layer was then dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 68 mg (48%) of compound 35. 1 H NMR (MeOD): 0.79 (s, 3H, 18-CH 3 ), 1.31-2.41 (residual CH and Cl-I 2 ), 2.97 (m, 2H, 6-CH 2 ), 4.16 (br s, IH, 17a-H), 6.59 (br s, 1H, CH=CH), 7.38 (d, 1H, CHar, J = 8.1 Hz), 7.46 (t, 1H, CHar, J = 7.7 Hz), 7.62 (d, IH, CHar, J = 7.8 Hz), 7.72 (m, 2H, CHar), 7.94 (s, 1H, CHar). 1002051 Synthesis of (16,173)-16-(3-carbamoylbenzyl)-17 hydroxyestra-1,3,5(10)-triene-3-carboxylic acid (36) [002061 To a solution of compound 35 (68 mg, 0.158 mmol) in EtOH (5 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (10 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 48 h. The resulting reaction mixture was filtered WO 2012/129673 PCT/CA2012/000316 - 71 on celite and then concentrated. The crude compound was purified by flash chromatography using DCM/MeOH (95:5) as the eluent system to provide 10 mg (15%) of compound 36. 'H NMR (MeOD): 0.92 (s, 3H, 18-CH 3 ), 1.16-2.49 (residual CH and CH 2 ), 2.88 (in, 2H, 6-CH 2 ), 3.17 (in, I H), 3.85 (br d, IH, 17a-H, J= 9.5 Hz), 7.41 (in, 3H, CHar), 7.71 (in, 3H), 7.76 (s, 1H, CHar); 1 3 C NMR (MeOD): 13.3, 27.2, 28.3, 30.4, 33.0, 38.8, 38.9, 39.3, 43.3, 45.4, 50.0, 83.0, 126.0, 126.4, 128.0, 129.1, 129.2, 129.4, 131.3, 133.5, 134.8, 138.0, 144.3, 143.6, 146.8, 171.0, 172.7. [00207] Synthesis of 3-1(16,170)-17-hydroxy-3 (hydroxymethyl)estra-1,3,5(10)-trien-16-yllmethyl}benzamide (37) 100208] To a solution of compound 36 (300 mg, 0.70 mmol) in anhydrous THF (20 mL), under an argon atmosphere, was successively added BOP (338 mg, 0.76 mmol) and DIPEA (145 pL, 0.84 mmol) at room temperature. The reaction mixture was stirred over a period of 10 min followed by the addition NaBH4 (30 mg, 0.79 mmol). The reaction mixture was then stirred for an additional hour, poured into water and extracted twice with EtOAc (2 x 30 mL). The combined organic layers were then washed with brine, dried with MgSO 4 and concentrated. The crude compound was subjected to consecutive purifications by flash chromatography using DCM/MeOH (95:5) as the eluent in the first purification and acetone/hexanes (1:1) as the eluant in the second purification to provide 88 mg (30%) of compound 37. 'H NMR (MeOD): 0.92 (s, 3H, 18-CH 3 ), 1.15-2.49 (residual CH and CH 2 ), 2.83 (in, 2H, 6-CH 2 ), 3.17 (in, I H), 3.85 (br d, IH, 17a-H, J = 9.5 Hz), 4.89 (s, 2H, CH20H), 7.03 (s, IH, CHar), 7.08 (d, 1H, CHar, J = 8.1 Hz), 7.27 (d, IH, CHar, J = 8.0 Hz), 7.41 (in, 2H, CHar), 7.69 (d, I H, CHar, J = 7.8 Hz), 7.76 (s, I H, CHar); 1 3 C NMR (MeOD): 13.3, 27.4, 28.6, 30.5, 33.0, 38.8, 39.0, 39.7, 43.4, 45.4, 45.8, 50.0, 65.1, 83.0, 125.5, 126.0, 126.3, 128.6, 129.1, 129.4, 133.5, 134.8, 137.6, 139.6, 140.6, 144.4, 172.7. 100209] Synthesis of 3-{[(16 ,17p)-3-(bromomethyl)-17-hydroxyestra 1,3,5(10)-trien-16-yllmethyllbenzamide (38) WO 2012/129673 PCT/CA2012/000316 - 72 1002101 To a solution of compound 37 (65 mg, 0.15 mmol) in DCM (7 mL) at 0 0 C was added triphenylphosphine (61 mg, 0.23 mmol) and carbon tetrabromide (77 mg, 0.23 mmol). The reaction mixture was then stirred at 0 0 C over a period of 5 h. The reaction mixture was subsequently poured into water and extracted with DCM (2 x 20 mL). The combined organic layers were then dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 97:3) to provide 45 mg (60 %) of compound 38. 1H NMR (Acetone d 6 ): 0.92 (s, 3H, 18-CH 3 ), 1.14-2.48 (residual CH and CH 2 ), 2.82 (m, 2H, 6-CH 2 ), 3.22 (m, 1H), 3.85 (m, 2H, 17a-H and OH), 4.58 (s, 2H, CH 2 Br), 6.6 (br s, IH,

CONH

2 ), 7.12 (s, 1H, CHar), 7.19 (d, I H, CHar, J = Hz), 7.29 (d, 1H, CHar, J = Hz), 7.35 (t, IH, J = Hz), 7.41 (d and br s under peak, IH of CHar and 1H of CONH 2 , J = Hz), 7.74 (d, I H, CHar, J = Hz), 7.84 (s, 1H, CHar). 100211] Synthesis of 3-([(16p,17p)-3-(aminomethyl)-17-hydroxyestra 1,3,5(10)-trien-16-yllmethyl}benzamide (39) [00212] To a solution of compound 38 (30 mg, 0.06 mmol) in anhydrous DMF (3 mL) was added sodium azide (12 mg, 0.18 mmol). The solution was then stirred at 60'C over a period of 3 h while under an argon atmosphere. The reaction mixture was subsequently poured into water and extracted twice with EtOAc. The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound (25 mg) was then dissolved in ethanol (3 mL). Palladium on charcoal (10%) (10 mg) was then added while under an argon atmosphere. The reaction vessel was then flushed three times with hydrogen and stirred over a period of 24 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography using DCM/MeOH (95:5) as the eluent system to provide 15 mg (58%) of compound 39. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.13-2.49 (residual CH and CH 2 ), 2.82 (m, 2H, 6-CH 2 ), 3.17 (m, I H), 3.84 (d, I H, 17a-H, J= 9.4 Hz), 4.41 (s, 2H, CH 2

NH

2 ), 7.01 (s, 1H, CHar), 7.25 (d, IH, CHar, J = 8.1 Hz), 7.33 (m, 2H, CHar), 7.69 (d, I H, CHar, J = 7.5 Hz), 7.76 (s, 1 H, CHar); 1 3 C NMR (MeOD): 13.3, 27.4, 28.5, 30.5, 33.0, 38.8, WO 2012/129673 PCT/CA2012/000316 - 73 39.0, 39.7, 43.4, 45.4, 45.7, 45.9, 50.0, 83.0, 126.0, 126.2, 126.7, 129.1, 129.2, 129.4, 133.5, 134.8, 138.0, 138.8, 140.7, 144.4, 172.7. 1002131 General procedure for N-alkylation of 3-{{(16 ,17 )-3 (bromomethyl-1 7-hydroxvestra-1,3,5(10)-trien-16-vllmethyllbenzamide (compounds 40a-d) [002141 To a solution of compound 38 (25 mg, 0.06 mmol) in DCM (3 mL) was added triethylamine (43 pL, 3.0 mmol) and the appropriate amine (3.0 mmol). The resulting reaction mixture was stirred at room temperature over a period of 3 h. The reaction mixture was then poured into water, extracted twice with DCM, filtered using a phase separator device (Biotage) and concentrated. The desired N alkylamine derivatives were isolated following purification by flash chromatography (DCM/MeOH 95:5 to 9:1). [00215] 3-f[(160,170)-3-[(dimethylamino)methyll-17-hydroxyestra 1,3,5(10)-trien-16-yllmethyllbenzamide (40a): Yield (10 mg, 43%); 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.49 (residual CH and CH 2 ), 2.24 (s, 6H,

CH

2

N(CH

3

)

2 ), 2.82 (in, 2H, 6-CH 2 ), 3.17 (m, IH), 3.41 (s, 3H, CH 2 N), 3.84 (d, IH, 17a-H, J = 9.4 Hz), 4.41 (s, 2H, CH 2 N), 7.00 (s, 1H, CHar), 7.05 (d, I H, CHar, J = 8.1 Hz), 7.26 (d, 1H, CHar, J = 8.1 Hz), 7.41 (in, 2H, CHar), 7.69 (d, IH, CHar, J = 7.5 Hz), 7.76 (s, 1H, CHar); 1C NMR (MeOD): 13.3, 27.3, 28.6, 30.5, 33.0, 38.8, 39.0, 39.6, 43.4, 45.1 (2x), 45.4, 45.8, 50.0, 64.6, 83.0, 126.0, 126.3, 128.0, 129.1, 129.4, 131.3, 133.5, 134.8, 135.4, 137.7, 140.9, 144.4, 172.7. 1002161 3-f (16B,178)-3-(dipropylamino)methyll-17-hydroxyestra 1, 3 ,5(10)-trien-16-yllmethyllbenzamide (40b): Yield (5 mg, 21%); IH NMR (MeOD): 0.88 (t, 3H, CH 3

CH

2

CH

2 N), J = 7.4 Hz), 0,92 (s, 3H, 18-CH 3 ), 1.27 (t, 3H,

CH

3

CH

2 N, J = 7.2 Hz), 1.15-2.65 (residual CH and CH 2 ), 2.82 (m, 2H, 6-CH 2 ), 3.17 (in, IH), 3.41 (s, 3H, CH 2 N), 3.58 (s, 2H, PhCH 2 N), 3.84 (d, 1H, 17a-H, J = 9.4 Hz), WO 2012/129673 PCT/CA2012/000316 - 74 4.41 (s, 2H, CH 2

NH

2 ), 7.01 (s, IH, CHar), 7.07 (d, 1H, CHar, J = 8.1 Hz), 7.26 (d, IH, CHar, J = 8.1 Hz), 7.41 (m, 2H, CHar), 7.70 (d, IH, CHar, J = 7.5 Hz), 7.76 (s, 1H, CHar); "C NMR (MeOD): 11.3, 12.1, 13.3, 20.1, 27.3, 28.6, 30.5, 33.0, 38.8, 39.0, 39.6, 43.4, 45.4, 45.8, 48.1, 50.0, 55.8, 58.8, 58.4, 83.0, 126.0, 126.3, 128.1, 129.1, 129.4, 131.3, 133.5, 134.8, 137.7, 140.8, 144.4, 172.7. 100217] 3-1(16p,17p)-17-hydroxy-3-(pyrrolidin-1-ylmethyl)estra 1,3,5(10)-trien-16-yllmethyllbenzamide (40c): Yield (7 mg, 28%); 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.15-2.65 (residual CH and CH 2 ), 2.82 (m, 2H, 6

CH

2 ), 3.17 (m, 1H), 3.57 (s, 3H, CH 2 N), 3.84 (d, I H, 17a-H, J = 9.4 Hz), 7.02 (s, 1H, CHar), 7.07 (d, IH, CHar, J = 8.1 Hz), 7.25 (d, 1H, CHar, J = 8.1 Hz), 7.41 (m, 2H, CHar), 7.71 (d, 1H, CHar, J = 7.5 Hz), 7.76 (s, 1H, CHar); "C NMR (MeOD): 11.9, 22.6, 25.9, 27.2, 29.1, 31.6, 37.4, 37.6, 38.2, 42.0, 44.0, 44.4, 47.0, 48.2, 48.6, 53.4, 59.7, 81.6, 124.6, 124.9, 126.4, 127.7, 128.0, 129.6, 132.1, 133.4, 134.6, 136.3, 139.4, 143.0, 171.3. [002181 3-{[(160,176)-17-hydroxy-3-(methylamino)methyllestra 1,3,5(10)-trien-16-yllmethyllbenzamide (40d): Yield (2 mg, 8%); 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.49 (residual CH and CH 2 ), 2.51 (s, 3H,

NHCH

3 ), 2.82 (m, 2H, 6-CH 2 ), 3.17 (m, IH), 3.84 (m, 2H, 17a-H and CH 2 NH), 7.07 (s, IH, CHar), 7.13 (d, IH, CHar, J = 8.3 Hz), 7.33 (d, 1H, CHar, J = 8.0 Hz), 7.40 (m, 2H, CHar), 7.69 (d, I H, CHar, J 7.5 Hz), 7.76 (s, IH, CHar). [002191 General procedure for N-acylation of (16E,173)-16-(3 carbamovlbenzylidene)-1 7-hydroxyestra-1,3,5(10)-triene-3-carboxylic acid (35) (compounds 41a-d) [002201 To a solution of compound 36 (50 mg, 0.12 mmol) in DMF (3 mL) was added BOP (43 pL, 0.14 mmol) the appropriate amine (0.36 mmol) and DIPEA (28 pL, 0.17 mmol). The resulting reaction mixture was stirred at room WO 2012/129673 PCT/CA2012/000316 - 75 temperature over a period of 2 h. The reaction mixture was then poured into water and extracted twice with EtOAc. The combined organic layers were washed with water, brine, dried with MgSO 4 and concentrated. The desired N-acylated derivatives were isolated following purification by flash chromatography (DCM/MeOH 95:5 to 9:1). 100221] (16 ,17 )-16-(3-carbamoylbenzvl)-17-hydroxy-NN dimethylestra-1,3,5(10)-triene-3-carboxamide (41a): Yield (7 mg, 13%); 1 H NMR (MeOD): 0.92 (s, 3H, 18-CH 3 ), 1.16-2.49 (residual CH and CH 2 ), 2.51 (s, 3H,

NHCH

3 ), 2.86 (m, 2H, 6-CH 2 ), 3.10 and 3.32 (2 s, 6H, (CH 3

)

2 NCO), 3.17 (m, 1H), 3.85 (in, 2H, 17a-H), 7.11 (s, I H, CHar), 7.17 (d, IH, CHar, J = 8.1 Hz), 7.33 (d, IH, CHar, J = 8.0 Hz), 7.41 (m, 2H, CHar), 7.70 (d, I H, CHar, J = 8.1 Hz), 7.76 (s, 1H, CHar); 13 C NMR (MeOD): 13.3, 30.4, 33.0, 35.6, 38.0, 38.8,_38.9, 39.4, 41.9, 43.4, 45.0, 45.4, 45.8 (d), 50.0, 83.0, 88.2, 125.3, (126.0 and 126.2 (d)), 126.5, 128.5, 129.1 and 129.2 (d), (129.4 and 129.5 (d)), (133.5 and 133.6 (d)), (134.3, 134.8 and 134.9 (t)), 138.3, (143.6, 143.7 and 143.8 (t)), 144.3, 172.6, 174.2. 1002221 (161,170)-16-(3-carbamoylbenzvl)-N-ethyl-17-hydroxy-N propylestra-1,3,5(10)-triene-3-carboxamide (41b): Yield (17 mg, 30%); 1H NMR (MeOD): 0.92 (s, 3H, 18-CH 3 ), 1.16-2.49 (residual CH and CH 2 ), 2.51 (s, 3H,

NHCH

3 ), 2.86 (in, 2H, 6-CH 2 ), 3.10 and 3.32 (2 s, 6H, (CH 3

)

2 NCO), 3.17 (m, IH), 3.85 (m, 2H, 17a-H), 7.11 (s, IH, CHar), 7.17 (d, I H, CHar, J = 8.1 Hz), 7.33 (d, IH, CHar, J = 8.0 Hz), 7.41 (in, 2H, CHar), 7.70 (d, IH, CHar, J = 8.1 Hz), 7.76 (s, IH, CHar); 13 C NMR (MeOD): 13.3, 21.8, 23.0, 27.2, 28.3, 30.4, 33.0, 38.8, 38.9, 45.4, 45.8, 50.0, 82.9, 88.2, 124.4 and 124.6 (d), 126.5, 127.7 and 127.9 (d), 129.1, 129.4, 133.5, 134.8, 135.2, 138.3, 143.3, 144.3, 172.6, 174.2. 1002231 3-{l(16 ,17 )-1 7 -hydroxy-3-(pyrrolidin-1-vlcarbonyl)estra 1, 3

,

5 (10)-trien-16-yllmethyllbenzamide (41c): Yield (17 mg, 30%); 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.15-2.49 (residual CH and CH 2 ), 2.66 (d, 2H,

CH

2 PhCONH 2 , J = 9.4 Hz), 2.85 (m, 2H, 6-CH 2 ), 3.17 (in, IH), 3.47 (t, 2H, CH 2 N of WO 2012/129673 PCT/CA2012/000316 - 76 pyrrolidine, J = 6.6 Hz), 3.58 (t, 2H, CH 2 N of pyrrolidine, J = 6.9 Hz), 3.84 (d, 2H, 17a-H, J = 9.4 Hz), 7.21 (s, IH, CHar), 7.26 (d, 1 H, CHar, J = 8.2 Hz), 7.40 (in, 2H, CHar), 7.69 (d, 1H, CHar, J = 8.1 Hz), 7.76 (s, IH, CHar); 13 C NMR (MeOD): 13.3, 25.3, 27.2 (2x), 28.3, 30.4, 33.0, 38.9, 39.4, 43.3, 45.4, 45.8, 50.0, 50.9, 82.0, 88.2, 125.3, 126.0, 126.4, 128.6, 129.4, 133,5, 134.8, 135.1, 138.2, 144.1, 144.3, 172.1, 172.6. 100224] (16 ,170)-1 6 -(3-carbamoylbenzyl)-17-hydroxy-N-methylestra 1, 3 ,5(10)-triene-3-carboxamide (41d): Yield (22 mg, 42%); 1H NMR (MeOD): 0.92 (s, 3H, 18-CH 3 ), 1.15-2.49 (residual CH and CH 2 ), 2.51 (s, 3H, NHCHI), 2.9 (m, 5H, 6-CH 2 and CH 3 NH), 3.17 (m, 1 H), 3.85 (m, 2H, 17a-H), 7.51 (s, 1H, CHar), 7.55 (d, IH, CHar, J = 8.2 Hz), 7.69 (d, 1H, CHar, J = 7.5 Hz), 7.76 (s, I H, CHar); 1 3 C NMR (MeOD): 13.3, 27.2, 28.3, 30.5, 33.0, 38.8, 38.9, 39.3, 43.3, 45.3, 45.9, 50.0, 82.9, 88.2, 125.4, 126.0, 126.5, 128.7, 129.1, 129.4, 132.6, 133.5, 134.8, 138.2, 144.3, 145.6, 170.9, 172.7. [002251 Synthesis of 3

-(

4

,

4 ,5,5-tetramethyl-1,3,2-dioxaborolan-2 yl)estra-1(10),2,4-trien-17-one (43) 100226] To a solution of estrone triflate (42) (455 mg, 1.12 mmol) in anhydrous dioxane (5 mL) at room temperature was added pinacolborane, Pd(dppf)C1 2 and triethylamine (923 pL, 6.62 mmol) under an argon atmosphere. The reaction mixture was bubbled with argon over a period of 5 min and then heated at 100 0 C over a period of 24 h. The dioxane was removed under reduce pressure followed by the addition of EtOAc (50 mL). The organic layer was washed successively with water and brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (EtOAc/Hexanes: 95:5 to 8:2) to provide 275 mg (64%) of compound 43. 'H NMR (CDCI 3 ): 0.91 (s, 3H, 18-CH 3 ), 1.34 (12H, 4 x CH 3 of borolan), 1.39-2.54 (residual CH and CH 2 ), 2.93 (m, 2H, 6

CH

2 ), 7.32 (d, IH, CHar, J = 7.8 Hz), 7.57 (s, 1H, CHar), 7.60 (d, IH, CHar, J = 7.9 Hz).

WO 2012/129673 PCT/CA2012/000316 - 77 1002271 Synthesis of [(16E)-16-(3-carbamoylbenzylidene)-17-oxoestra 1(10),2,4-trien-3-yllboronic acid (44) [002281 To a solution of compound 43 (150 mg, 0.39 mmol) in EtOH (10 mL) was added 3-formyl-benzamide (118 mg, 0.79 mmol) and aqueous KOH solution (10%, 1.5 mL). The reaction mixture was then heated at reflux over a period of 30 min. The resulting reaction mixture was then diluted with water (200 mL), neutralized with an aqueous HCl solution (10%), and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated to provide 80 mg of compound 44. 1H NMR (DMSO-d 6 ): 0.94 (s, 3H, 18-CH 3 ), 1.24 2.97 (residual CH and CH 2 ), 4.05 (s, 2H, B(OH)2, 7.27 (d, I H, CHar, J = 7.8 Hz), 7.37 (s, IH, CHar), 7.49-7.58 (in, 3H, CHar), 7.81 (d, IH, CHar, J = 7.7 Hz), 7.90 (d, I H, CHar, J = 7.8 Hz), 8.1 (s, 1H, CHar). 1002291 Synthesis of [(16E,17p)-16-(3-carbamoylbenzylidene)-17 hydroxyestra-1(10),2,4-trien-3-yllboronic acid (45) [002301 To a solution of compound 44 (70 mg, 0.16 mmol) in MeOH (6 mL) was added NaBH4 (10 mg, 0.26 mmol). The reaction mixture was stirred at room temperature over a period of 1h and concentrated. The residue was diluted with EtOAc (25 mL) and washed with water. The organic layer was then washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 9:1) to provide 54 mg (77 %) of compound 45. 'H NMR (MeOD): 0.77 (s, 3H, 18-CH 3 ), 1.24-2.97 (residual CH and CH 2 ), 4.05 (s, 2H, B(OH)2, 7.27 (d, IH, CHar, J = 7.8 Hz), 7.37 (s, IH, CHar), 7.49-7.58 (in, 3H, CHar), 7.81 (d, 1 H, CHar, J = 7.7 Hz), 7.90 (d, I H, CHar, J = 7.8 Hz), 8.1 (s, 1H, CHar). 100231] Synthesis of [(16p,17 )-16-(3-carbamoylbenzyl)-17 hydroxyestra-1(10),2,4-trien-3-yllboronic acid (46) WO 2012/129673 PCT/CA2012/000316 - 78 [00232] To a solution of compound 45 (47 mg, 0.11 mmol) in EtOH (4 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (8 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 24 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography using DCM/MeOH (95:5) as the eluent system to provide 13 mg (28%) of compound 46. 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.49 (residual CH and CH 2 ), 2.82 (in, 2H, 6-CH 2 ), 3.17 (in, 1H), 3.84 (d, 1H, 17-H, J = 9.4 Hz), 7.291 (in, 2H, CHar), 7.35-7.43 (in, IH, CHar), 7.70 (d, IH, CHar, J = 7.4 Hz), 7.76 (s, 1H, CHar); 1 3 C NMR (MeOD): 13.3, 27.2, 28.6, 30.5, 33.0, 38.8, 39.0, 39.6, 43.4, 46.0, 50.0, 83.0, 125.4 and 125.5 (d), 126.0, 129.1, 129.4, 131.9 and 132.2 (d), 133.5, 134.8, 135.4 and 135.7 (d), 143.0 and 143.7, 144.4, 172.7. [002331 Synthesis of 3 -{{E)-[(16E)-3-amino-17-oxoestra-1,3,5(10)-trien 16-ylidenelmethyllbenzamide (48) [00234] To a solution of 3-amino-estrone (47) (68 mg, 0.25 mmol) in EtOH (3.5 mL) was added 3-formyl-benzamide (75 mg, 0.50 mmol) and aqueous KOH solution (10%, 0.5 mL). The reaction mixture was then heated at reflux over a period of 1 h. The resulting reaction mixture was then diluted with water (50 mL), neutralized with an aqueous HCl solution (10%), and extracted with EtOAc (3 x 15 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography using DCM/MeOH (95:5) as the eluent system to provide 40 mg (40%) of compound 48. 'H NMR (Acetone-d 6 ): 1.00 (s, 3H, 18-CH 3 ), 1.29-3.06 (residual CH and CH 2 ), 4.33 (br s, 2H, PhNH 2 ), 6.40 (s, I H, CHar), 6.47 (d, IH, CHar, J = 8.3 Hz), 7.00 (d, 1 H, CHar, J = 8.3 Hz), 7.40 (s, 1H, CHar), 7.57 (t, I H, CHar, J = 7.7 Hz), 7.83 (d, I H, CHar, J 7.8 Hz), 7.96 (d, 1 H, J = 7.8 Hz), 8.20 (s, I H, CHar). [002351 Synthesis of 3-{(E)-[(16E,170)-3-amino-17-hydroxyestra 1,3,5(10)-trien-16-ylidenelmethyl}benzamide (49) WO 2012/129673 PCT/CA2012/000316 - 79 [002361 To a solution of compound 48 (40 mg, 0.10 mmol) in MeOH (6 mL) was added NaBH 4 (19 mg, 0.50 mmol). The reaction mixture was stirred at room temperature over a period of lh and concentrated. The residue was diluted with EtOAc (25 mL) and washed with water. The organic layer was then washed with brine, dried with Na 2

SO

4 and concentrated. The crude compound was purified by flash chromatography (DCM/MeOH: 95:5) to provide 26 mg (65%) of compound 49. 1H NMR (MeOD): 0.77 (s, 3H, 18-CH 3 ), 1.29-2.82 (residual CH and CH 2 ), 4.13 (s. I H, 17a-H), 6.50 (s, I H, CHar), 6.56 (m, 2H, CHar), 7.07 (d, 1 H, CHar, J = 8.0 Hz), 7.46 (t, IH, CHar, J = 7.7 Hz), 7.60 (d I H, CHar, J = 7.8 Hz), 7.69 (d, IH, CHar, J = 7.9 Hz), 7.94 (s, I H). [00237] Synthesis of 3-f{(16 ,17 )-3-amino-17-hydroxyestra-1,3,5(10) trien-16-yllmethyllbenzamide (50) and 3-{[(160,170)-3-(ethylamino)-17 hydroxyestra-1,3,5(10)-trien-16-yljmethyl}benzamide (51) [002381 To a solution of compound 49 (15 mg, 0.04 mmol) in EtOH (2 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (5 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 24 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography using Acetone/Hexanes (1:1) as the eluent system to provide 4 mg (27%) of compound 50 and 5 mg (33%) of compound 51. 1002391 Compound 50: 'H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14 2.85 (residual CH and CH 2 ), 3.83 (d, I H, 17ax-H, J = 9.4 Hz), 6.46 (s, lH, CHar), 6.55 (d, 1H, CHar, J = 8.3 Hz), 7.04 (d, 1H, CHar, J = 8.5 Hz), 7.41 (m, 2H, CHar), 7.69 (d, 1H, CHar, J = 7.4 Hz), 7.75 (s, 1H); "C NMR (MeOD): 13.3, 27.5, 28.7, 30.7, 33.0, 38.9, 39.0, 40.0, 43.4, 45.5, 50.0, 83.1, 88.4, 114.9, 117.2, 126.0, 126.8, 129.1, 129.4, 132.0, 133.5, 134.8, 138.2, 144.4, 145.5, 172.7.

WO 2012/129673 PCT/CA2012/000316 - 80 [002401 Compound 51: 1H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.22 (t, 3H, CH 3

CH

2 NH, J = 7.1 Hz), 1.12-2.48 (residual CH and CH 2 ), 2.74 (2H, 6-CH 2 ), 3.08 (q, 2H, CH 3

CH

2 NH, J = 7.1 Hz), 3.17 (m, 1H), 3.83 (d, I H, 17at-H, J = 9.4 Hz), 6.39 (s, IH, CHar), 6.49 (d, IH, CHar, J = 8.3 Hz), 7.06 (d, 1H, CHar, J = 8.5 Hz), 7.41 (in, 2H, CHar), 7.69 (d, 1H, CHar, J 7.4 Hz), 7.76 (s, 1H); 1 3 C NMR (MeOD): 13.4, 14.9, 27.6, 28.8, 30.9, 33.0, 38.9, 39.0, 40.0, 40.1, 43.4, 45.5, 50.0, 51.7, 83.1, 113.0, 115.0, 126.0, 126.2, 129.1, 129.4, 131.1, 133.5, 134.8, 138.2, 144.4, 147.8, 167.7. [00241] Synthesis of 3-fl(16@,17 )-17-hydroxy-3-(methylamino)estra 1,3,5(10)-trien-16-yllmethyllbenzamide (53) [002421 To a solution of compound 49 (22 mg, 0.05 mmol) in EtOH (3 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (5 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 24 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by preparative chromatography using EtOAc/Hexanes (1:1) as the eluent system to provide 3 mg (14%) of compound 50 and 6 mg (27%) of compound 53. 100243] Compound 53: 1H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.13 2.48 (residual CH and CH 2 ), 2.74 (5H, 6-CH 2 and CH 3 NH), 3.17 (in, IH), 3.83 (d, 1H, 17a-H, J = 9.4 Hz), 6.39 (s, IH, CHar), 6.47 (d, IH, CHar, J = 8.3 Hz), 7.07 (d, IH, CHar, J = 8.5 Hz), 7.41 (m, 2H, CHar), 7.69 (d, 1H, CHar, J = 7.4 Hz), 7.76 (s, 1H); 3 C NMR (MeOD): 13.4, 27.6, 28.8, 30.9, 31.3, 33.0, 35.2, 38.9, 39.0, 40.1, 43.4, 45.5, 50.0, 83.1, 112.4, 114.2, 126.0, 126.8, 129.1, 129.4, 130.9, 133.5, 134.8, 138.1, 144.4, 148.9, 151.1, 167.7. [002441 Synthesis of 3- {E)-I(16E)-3-fluoro-1 7 -oxoestra-1(10),2,4-trien 16-ylideneimethyll benzamide (55) WO 2012/129673 PCT/CA2012/000316 -81 [002451 To a solution of 3-fluoro-estrone (54) (169 mg, 0.62 mmol) in EtOH (10 mL) was added 3-formyl-benzamide (175 mg, 1.17 mmol) and aqueous KOH solution (10%, 1.7 mL). The reaction mixture was then heated at reflux over a period of I h. The resulting reaction mixture was then diluted with water (100 mL), neutralized with an aqueous HCI solution (10%), and extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography using EtOAc/Hexanes (1:1) as the eluent system to provide 146 mg (62%) of compound 55. H NMR (MeOD): 1.05 (s, 3H, 18-CH 3 ), 1.23-2.76 (residual CH and CH 2 ), 6.84 (in, 2H, CHar), 7.32 (t, IH, CHar, J = 7.7 Hz), 7.49 (s, IH, CHar), 7.59 (t, IH, CHar, J 7.8 Hz), 7.82 (d, I H, CHar, J = 7.8 Hz), 7.92 (d, I H, J = 7.7 Hz), 8.14 (s, 1H, CHar). [002461 Synthesis of 3-{(E)-[(16E,17p)-3-fluoro-17-hydroxyestra 1(10),2,4-trien-16-ylidenelmethyllbenzamide (56) [002471 To a solution of compound 55 (140 mg, 0.35 mmol) in a mixture of MeOH/DCM (2:1) (15 mL) was added NaBH4 (26 mg, 0.68 mmol). The reaction mixture was stirred at room temperature over a period of 30 min and concentrated. The residue was diluted with DCM (50 mL) and washed with water. The organic layer was then washed with brine, dried with Na 2

SO

4 and concentrated. The crude compound was triturated with diethyl ether and filtered to provide 140 mg (99%) of compound 56. 'H NMR (MeOD): 0.77 (s, 3H, 18-CH 3 ), 1.30-2.82 (residual CH and

CH

2 ), 2.92 (in, 2H, 6-CH 2 ), 4.13 (s, I H, 17a-H), 6.58 (s, IH, CHar), 6.82 (in, 2H, CHar), 7.30 (m, IH, CHar), 7.45 (t, IH, CHar, J = 7.7 Hz), 7.58 (d, I H, CHar, J = 7.9 Hz), 7.69 (d, I H, CHar, J = 7.8 Hz), 7.94 (s, 1H, CHar). [00248] Synthesis of 3-{[(16 ,17B)-3-fluoro-17-hydroxyestra-1(10),2,4 trien-16-Yl methyl}benzamide (57) WO 2012/129673 PCT/CA2012/000316 - 82 1002491 To a solution of compound 56 (140 mg, 0.35 mmol) in EtOH (5 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (28 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 48 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography using Acetone/Hexanes (6:4) as the eluent system to provide 59 mg (42%) of compound 57. 1H NMR (MeOD): 0.91 (s, 3H, 18-CH 3 ), 1.14-2.62 (residual CH and CH 2 ), 2.82 (in, 2H, 6-CH 2 ), 3.17 (in, IH), 3.84 (d, 1H, 17ax-H, J = 9.4 Hz), 6.78 (in, 2H, CHar), 7.07 (d, I H, CHar, J = 8.5 Hz), 7.30 (in, IH, CHar), 7.41 (in, 2H, CHar), 7.69 (d, 1H, CHar, J = 7.4 Hz), 7.76 (s, 1H); "C NMR (MeOD): 13.3, 27.4, 28.3, 30.5, 33.0, 38.8, 39.4, 43.3, 45.3, 49.8, 82.9, 88.2, 113.0 and 113.2 (d), 115.6 and 115.8 (d), 125.9, 127.9 (d), 129.1, 129.4, 133.5, 134.7, 137.3, 140.1 (d), 144.3, 161.0 and 163.4 (d), 172.6. 100250] Synthesis of (16E)-16-(3-carbamoylbenzylidene)-17-oxoestra 1,3,5(10)-triene-3-carboxamide (59) 100251] To a solution of 3-carboxamide-estrone (58) (250 mg, 0.84 mmol) in EtOH (10 mL) was added 3-formyl-benzamide (250 mg, 1.67 mmol) and aqueous KOH solution (10%, 1.7 mL). The reaction mixture was then heated at reflux over a period of 40 min. The resulting reaction mixture was then diluted with water (150 mL), neutralized with an aqueous HCI solution (10%), and extracted with EtOAc (3 x 35 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography using DCM/MeOH (95:5) as the eluent system to provide 70 mg (19%) of compound 59. 'H NMR (DMSO): 0.95 (s, 3H, 18-CH 3 ), 1.43-2.74 (residual CH and CH 2 ), 2.95 (in, 3H), 7.24 and 7.50 (2 s, 2H, CONH 2 ), 7.37 (d, 2H, CHar, J = 7.6 Hz), 7.57 (t, IH, CHar, J = 7.7 Hz), 7.63 (d, 2H, CHar, J = 9.7 Hz), 7.82 (d, I H, CHar, J = 7.9 Hz), 7.87 and 8.07 (2s, 2H, CONH 2 ), 7.90 (d, 1H, J = 7.8 Hz), 8.10 (s, 1H, CHar).

WO 2012/129673 PCT/CA2012/000316 - 83 [002521 Synthesis of (16E,17p)-16-(3-carbamoylbenzvlidene)-17 hydroxyestra-1,3,5(10)-triene-3-carboxamide (60) 1002531 To a solution of compound 59 (68 mg, 0.16 mmol) in a mixture of MeOH/DCM (1:1) (30 mL) was added NaBH4 (10 mg, 0.26 mmol). The reaction mixture was stirred at room temperature over a period of 1 h and concentrated. The residue was diluted with DCM (50 mL) and washed with water. The organic layer was then washed with brine, dried with Na 2

SO

4 and concentrated. The crude compound was triturated with diethyl ether and filtered to provide 68 mg (99%) of compound 60. 1H NMR (MeOD): 0.78 (s, 3H, 18-CH 3 ), 1.43-2.86 (residual CH and

CH

2 ), 3.01 (in, 2H, 6-CH 2 ), 4.15 (s, IH, 17a-H), 6.59 (s, 1H, CHar), 7.47 (in, 2H, CHar), 7.62 (in, 3H, CHar), 7.69 (d, I H, CHar, J = 7.8 Hz), 7.95 (s, I H, CHar). 1002541 Synthesis of (160,17)-16-(3-carbamoylbenzvl)-17 hydroxyestra-1,3,5(10)-triene-3-carboxamide (61) [00255] To a solution of compound 60 (64 mg, 0.148 mmol) in EtOH (5 mL) at room temperature and under an argon atmosphere was added palladium on charcoal (10%) (11 mg). The reaction vessel was then flushed three times with hydrogen and stirred over a period of 16 h. The resulting reaction mixture was filtered on celite and then concentrated. The crude compound was purified by flash chromatography usingDCM/MeOD (95:5) as the fluent system to provide 21 mg (32%) of compound 61. 'H NMR (MeOD): 0.93 (s, 3H, 18-CH 3 ), 1.17-2.49 (residual CH and CH 2 ), 2.89 (in, 2H, 6-CH 2 ), 3.18 (in, 1H), 3.86 (d, lH, l7c-H, J = 9.5 Hz), 7.40 (in, 3H, CHar), 7.58 (s, I H, CHar), 7.62 (d, 1 H, CHar, J = 8.2 Hz), 7.69 (d, I H, CHar, J = 6.1 Hz), 7.76 (s, I H). "C NMR (MeOD): 11.9, 25.8, 26.9, 29.0, 31.6, 37.4, 37.5, 37.9, 41.9, 43.9, 44.5, 48.6, 81.5, 124.5, 124.6, 125.1, 127.7, 127.8, 128.0, 130.5, 132.1, 133.4, 136.7, 142.9, 144.6, 171.2. [00256] 170-HSD3 WO 2012/129673 PCT/CA2012/000316 - 84 1002571 The 17p-HSD3 enzyme represents a promising target for the treatment of advanced prostate cancer by blocking the different sources of active androgen T from the testis (endocrine), adrenals (intracrine) and intratumoral tissues (intracrine) (FIG. 10). Furthermore, it is believed that the use of 17p-HSD3 inhibitors complementary to actual endocrine therapy could increase the efficiency of the antiandrogens, LHRH agonists, and inhibitors of androgen biosynthesis enzymes (5a reductase and 17a-lyase) by providing a synergistic effect.

44

'

45 This more complete inactivation of the androgen receptor (AR) could also limit its ability to mutate, and/or diminish the ability of androgen-independent clones to evolve toward androgen independence. [002581 Previous structure-activity relationship (SAR) studies on the inhibition of 17p-HSD3 have identified important criteria for inhibitory activity (FIG. 11A).46-5 Despite of their good efficiency in homogenated HEK-293 cells overexpressing 170-HSD3, these inhibitors were however not optimal. The first generation inhibitors of the 3p-alkyl-androsterone series showed an androgenic profile on Shionogi (ARt) cells which is undesirable in the context of a treatment for an androgen-sensitive disease. The inhibitory potency of these inhibitors was also lower in intact than in homogenated HEK-293 cells overexpressing 17p-HSD3, thus suggesting low cell permeability. 100259] To provide 17p-HSD3 inhibitors with a non-androgenic profile, the enzyme's tolerance for new diversified substituents introduced at position 3 of androsterone (ADT) nucleus was explored. More specifically, two series of ADT derivatives (tertiary amines and carbamates) were designed, synthesized and tested as new inhibitors of 17p-HSD3 (FIG. 11B). [002601 The ADT derivatives 7a-j were readily obtained in acceptable yields (35% to 70%) from the ring opening of oxirane 6 in refluxing ethanol using a series of commercially available secondary amines (Scheme 15). Similarly, oxirane 6 was also opened with piperazine or trans-2,5-dimethylpiperazine to provide WO 2012/129673 PCT/CA2012/000316 - 85 intermediates 8 and 9. The free NH of 8 and 9 was subsequently submitted to a series of addition reactions using a variety of building blocks (e.g. benzyl bromide, acyl chloride and sulfonyl chloride) to obtain the corresponding piperazine derivatives 1Oa-j and lla-j (amines); 12a-e and 13a-d (amides); and 14a-c and 15a-c (sulfonamides). 0 0 H a H H (see Table 1 R 0 6 for Ri and R2) R2 H 7a-j 0 b c R N Rl -N HO (see Table 2 R for R) 10 a-j (R = H) 0 11 a-j (R = CH 3 ) 0 Rd a R N R N~ N - (see Table 2 R N HO HN RHO foI4 R R HforR) 0 12a-e (R = H) 8 (R = H) 13a-d (R = CH 3 ) O 9 (R = CH 3 ) e (see Table 2 R1__ N HO forR 1 ) R 14a-c (R = H) 15a-c (R = CH 3 ) Scheme 15: Reagents and conditions: (a) R'R 2 NH, ethanol, 60'C; (b) piperazine or 2,5 dimethylpiperazine, ethanol, 60'C; (c) R'CH 2 Br; TEA, DCM, rt; (d) R'COCI, TEA, DCM, rt; (e) R'SO 2 C, TEA, DCM. [00261] The carbamate derivatives 17a-i, 18, 21, 22 and 23a-g were prepared as illustrated hereinbelow in Schemes 16 and 17. Carbamates 17a-i and 18 were prepared from the ring opening of oxirane 6 in refluxing ethanol using a series of commercially available primary amines followed by a cyclization reaction WO 2012/129673 PCT/CA2012/000316 - 86 converting the amino-alcohol into a carbamate. Finally, C16 di-methylation of 17b provided 18 (Scheme 16). 0 0 a b R-N" O 6 H Hd 16 0 0 C R-N 17N O17 0 0~ ~ 0 18 R =

OCH

3 (17a) (17b) (17c) _ CF 3 N CF 3 1F 3 C (17d) (17e) (17f)

CF

3

CF

3

CF

3 (17fg (17h) (17i) Scheme 16: Reagents and conditions: (a) R-NH 2 , ethanol, 60'C; (b) triphosgene, DIPEA, DCM; (c) NaH, Mel, DMF. [00262] Carbamates 21, 22 and 23a-g were prepared from the ring opening of oxirane 6 in refluxing ethanol using aminoethanol-O-TBDMS followed by a cyclization reaction converting the amino-alcohol 19 into carbamate 20. Removal of the TBDMS protecting group provided alcohol 21 which was readily transformed into the corresponding bromide 22. Finally, carbamates 23a-g were obtained by different N, 0 and S-alkylation reactions of the key alkyl bromide intermediate 22 (Scheme 17).

WO 2012/129673 PCT/CA2012/000316 -87 0 0 b a , TBDMSON b 6 HIH 6 19 0 0 C d TBDMSO - \ HO N 0 -O O 20 0 21 0 0 e,f,gorh Br---/'- tR OO 00 22 23 = 0N--

N

0 (23a) (23b) (23c) (23d) \0 N-- N

-

(23e) (23f) (23g) Scheme 17: Reagents and conditions: (a) NH 2

(CH

2

)

2 OTBDMS, ethanol, 60 0 C; (b) triphosgene, DIPEA, DCM; (c) TBAF, THF, rt; (d) PPh 3 , CBr 4 , DCM, 0 0 C to rt; (e) 3 methylphenol (for 23a); (f) i) 3-methylthiophenol,

K

2

CO

3 , DMF, 80'C; ii) KHSO 5 , MeOH/H 2 0 (for 23b); (g) appropriate R'R 2 NH, ethanol 60'C (for 23c, 23d, 23e and 23f); (h) i) propylamine, Na 2

CO

3 , DMF, 80'C; ii) propionyl chloride, triethylamine, DCM, rt (for 23g). [002631 Inhibition of 1 7fl-HSD3 in homogenized cells [002641 Tertiary Amine Derivatives [00265] Previous SAR studies showed that a hydrophobic chain at position 3p of ADT was well tolerated by 170-HSD3. Unfortunately, these compounds were WO 2012/129673 PCT/CA2012/000316 - 88 also found to stimulate the proliferation of androgen-sensitive cells and to possess relatively high hydrophobicity as illustrated in FIG. 11B with phenylpropyl-ADT (1). In view of their lower hydrophobic character as well as their potential for structural diversification, various tertiary amine derivatives were examined.s 3 The ADT scaffold was modified at position 3p using various acyclic (7a-d) and cyclic (7e-7j) amines and the resulting compounds were evaluated for their potential as inhibitors of 17p HSD3 (Table 2). [00266] Table 2: Inhibition of 17p-HSD3 in homogenized and intact cells by various tertiary-amine derivatives of ADT (compounds 7a-j),a 0 H R : OH H Homo Homo Homo Intact cells Intact cells Compound R (%) (%) (%) (%) (%) (0.01 pIM) (0.1 pIM) (I IM) (0.1 PM) ( pM) Ph-\ 7a N--- 42 85 90 86 94 7b N-- 28 79 89 48 92 7c N--- 0 10 60 N/A 14 7d N-- 13 70 85 53 90 7e N--- 9 19 69 N/A 23 7f N--- 40 82 90 N/A 52 7g N N--- 0 4 69 N/A 20 7h -N N--- 0 0 40 2 N/A 7i Ph-N N--- 13 78 89 N/A 68 .j Ph

-

7j -N N-- 35 72 90 37 80 WO 2012/129673 PCT/CA2012/000316 - 89 a For the transformation of [ 14 C]-4-androstene-3,17-dione (50 nM) into [ 1 4 C]-testosterone at the indicated concentration of tested compound. [00267] The ability of compounds 7a-7j to inhibit 17p-HSD3 transfected in human embryonic kidney (HEK)-293 cells (homogenized) was determined by measuring the amount of labelled T formed from natural labelled substrate 4 -dione in the presence of NADPH as cofactor. The results were expressed as the percent of inhibitory activity for a given compound. In the series of acyclic derivatives, compound 7a was the most active inhibitor (42% at 0.01 pM and 85% at 0.1 pM). For the cyclic derivatives, compounds 7f and 7j were the best inhibitors with 40% and 35% inhibition at 0.01 pM, respectively. Piperazine derivative 7j was however considered more interesting considering its lower hydrophobicity (compared to 7f). [00268] Piperazine Derivatives [00269] Various piperazine derivatives were prepared and analyzed (Table 3). The inhibition values remained substantially unchanged for various 3p-piperazine ADT derivatives comprising a substituted aryl group (Z) attached to the piperazine moiety (compounds 10a-j). Furthermore, the presence of either electron withdrawing groups (e.g. CF 3 ; Cl) or electron donating groups (e.g. OCH 3 ) had little or no effect on the inhibition values. Compound 10a demonstrated better inhibition than reference compound 7j. The pyridine ring (compound 10g) appears to have a negative impact on the inhibition of 17p-HSD3, indicative of an apparently poor tolerance for polar substituents. Yet furthermore, modifications to the linker (Y) (e.g. CH 2 , CO or SO 2 ) had little or no effect on the inhibitory activity (the rest of the compound remaining unchanged). Although the use of a trans-2,5-dimethylpiperazine moiety did not increase the inhibition in the amine (Y = CH 2 ) or amide (Y = CO) series of compounds, when compared to the corresponding compounds comprising a piperazine moiety, it did increase the inhibition in the sulfonamide series. Inhibition values of 55% and 79% at 0.01 piM were recorded for compounds 15b and 15c respectively. Sulfonamide 15c provided a higher percentage of inhibition (79% at WO 2012/129673 PCT/CA2012/000316 - 90 0.01 VtM) than its corresponding amide 13d (17%) and its corresponding amine l1c (14%). A strong negative impact on inhibition could also be observed when the C19 steroid nucleus was substituted for the C2 1-steroid nucleus which is indicative of 17p HSD3 having a clear preference for the androstane scaffold compared to the pregnane scaffold (84% inhibition at 0.1 pM vs. 12% inhibition respectively when Z = C 6

H

5 ; Y = CH 2 and X = piperazine). Finally, the presence of an insaturation at positions C4-C5 of the androstane derivative 7j had no significant impact on enzyme inhibition. [00270] Table 3: Inhibition of 17p-HSD3 in homogenized and intact cells: optimization of 3p-piperazine ADT series (compounds lOa-j, lla-j, 12a-e, 13a-d, 14 a-c and 15a-c).a 0 H Z-Y-X" H H OH H Compound Z Y X Homo (%) Homo (%) Intact cells (%) (0.01 jIM) (0.1 pM) (0.1 PM) 7j / -- CH 2 ---N N--- 36 74 38 1Oa CH 2 --- N N--- 51 88 52

F

3 C 10b - CH 2 --- N N--- 18 86 20

CF

3 lOc FC -- CH 2 -- N N--- 14 81 33 10d - CH 2 --- N N--- 3 91 N/A (62)b

F

3 CS loe CH 2 ---N N--- 26 85 N/A ( 7 5 )b CI ]Of - - CH 2 --- N N--- 21 83 47

H

3 CO log - CH 2 ---N N--- 10 33 N/A (63)b

CF

3 10h -- CH 2 ---N N--- 18 85 12

F

3

C

WO 2012/129673 PCT/CA2012/000316 -91 loi F 3 C_ CH 2 ---N N--- 22 75 16

F

3 C F3C 1Oj CH 2 ---N N--- 25 83 25

F

3 C 1a - CH 2 -- N N-- 31 89 N/A(78)

F

3 C /N \ Ilb - CH 2 -- N N-- 22 85 36

CF

3 1lc F 3 C CH 2 -- N N-- 14 87 36 lid CH 2 -- N N-- 18 88 N/A (58)

F

3 CS lie CH 2 -- N N-- 26 84 N/A (55)' CI 11f CH 2 -- N N-- 19 87 50

H

3 CO 1Ig - CH 2 -- N N-- 17 47 N/A (58)'

N

CF

3 11h -- CH 2 -- N N-- 16 71 23

F

3 C

CF

3 1li F 3 C -- 2 -- N N-- 15 71 21

F

3 C lj -- CH 2 -- N N-- 25 74 16

F

3 C 12a CO - -N N- - 17 76 50 12b -- CO --- N N--- 19 75 N/A (70)b 12c CO ---N N--- 31 86 62

CF

3 12d CO --- N N--- 10 92 91

F

3 C 12e F 3 C -- CO --- N N--- 22 84 56 13a CO -- N N-- 24 87 78 WO 2012/129673 PCT/CA2012/000316 - 92 13b -- CO -- N N-- 24 90 77 13c CO -- N N-- 28 89 70

F

3 C 13d FC/ -- CO -- N N-- 17 91 61 14a

SO

2 -- N N-- 21 82 N/A (71)b

F

3 C 14b S02 ---N N--- 45 92 63

CF

3 14c FC/ -- S02 ---N N--- 21 78 34 15a S02 -- N N-- 32 91 77

F

3 C 15b SO 2 -- N N-- 55 92 84

CF

3 15c FaC/ - SO 2 -- N N-- 79 92 47 "For the transformation of [ 14 C]-4-androstene-3,17-dione (50 nM) into [ 14 C]-testosterone at the indicated concentration of tested compound. 'Tested at 1 pM. [002711 Carbamate Derivatives 1002721 A small library of 3p-carbamate-ADT derivatives showing 46 promising inhibitory activities on 17p-HSD3 was previously synthesized. Despite the good activities, the compounds possess an ester group (see 4 and 5 in FIG. 11B) vulnerable to in vivo hydrolysis. In order to provide improved inhibitors with a more stable substituent on the carbamate moiety, a further series of new carbamate analogues was prepared (Table 4). [00273] Table 4: Inhibition of 170-HSD3 in homogenized and intact cells: optimization of 3-carbamate ADT series (compounds 17a-i, 18, 21, 22 and 23a-g).a WO 2012/129673 PCT/CA2012/000316 - 93 0 R -N OR 0 Compound R R2 Homo (%) Homo (%) Homo (%) Intact cells (%) (0.01 pM) (0.1 pM) (1.0 pM) (0.1 PM) 0 4 H 2 79 94 49 17a H 21 86 93 44 17b H 17 81 93 38 17c H3Co H 5 84 93 43 17d Fa H 25 80 nd 43 17e F 7c H 3 65 nd 36

F

3 C N 17f ff " H 17 81 nd 35

CF

3 17gF 3 C H 16 80 nd 36 17h Fac H 12 66 nd 35

CF

3 17i -- H 13 75 78 29 18 CH 3 2 22 74 18 21 H 0 9 38 16b 22 Br H 17 69 93 31 23a H 13 85 95 40 23b H 0 52 93 5 23c c N-x'. H 0 0 21 10h 23d H 14 71 93 40 23e N H 2 21 72 2 WO 2012/129673 PCT/CA2012/000316 -94 23f N H 0 10 62 8 23g H 0 35 80 25 "For the transformation of [ 1 4 C]-4-androstene-3,17-dione (50 nM) into [ 14 C]-testosterone at the indicated concentration of tested compound. "Tested in another experiment. [002741 The enzyme displayed good tolerance for all carbamates bearing a hydrophobic chain as represented by compounds 17a, 17b, 17d, 17f, 17g, 17h, 17i, 22 and 23a. Weak inhibition results were obtained for the carbamates bearing a hydrophilic chain as represented by compounds 17e, 21, 23b, 23c, 23e, 23f and 23g. This is particularly evident when the inhibitory activity of alcohol 21 is compared with the corresponding bromide 22, and the inhibitory activity of p-CF 3 -phenyl derivative 17d with p-CF 3 -pyridine derivative 17e. The sulfone derivative 23b was also less potent than its corresponding ether 23a. The addition of two methyl groups at position C16 of compound 17b was clearly not well tolerated by the enzyme and, consequently, compound 18 displayed only very weak inhibition of 17p-HSD3. A slight improvement of the inhibitory activity was observed for compounds 17a, 17b, 17d, 17f, 17g and 23a relative to reference compound 4. 100275] In one embodiment of the present disclosure, there are included inhibitors of 17B-HSD3, in which the inhibitor has the structure 0 H R OH H wherein R is heterocyclyl or -NRaRb, wherein Ra and Rb are each independently alkyl or aralkyl, or a pharmaceutically acceptable salt or tautomer thereof.

WO 2012/129673 PCT/CA2012/000316 - 95 100276] In another embodiment, there are included further inhibitors of 17B-HSD3, having the structure: 0 H R R Z-Y-X OH H wherein: -N N -- N N- X is or Y is -CH 2 -, -C(O)- or -S(O) 2 -; and Z is cycloalkyl, aryl or heterocyclyl, or a pharmaceutically acceptable salt or tautomer thereof. [002771 In one embodiment, the 17B-HSD3 inhibitor has the structure /0 H S-N N. - 0 OH H

CF

3 or a pharmaceutically acceptable salt thereof. In one embodiment, the inhibitor is the corresponding hydrochloride (HCI) salt of the above compound, or other acid addition salt on the piperazine moiety of the above compound.

WO 2012/129673 PCT/CA2012/000316 - 96 1002781 In another embodiment, the 17B-HSD3 inhibitor has the following prodrug structure OCOR H 0 S -N N .

CF

3 1002791 or a pharmaceutically acceptable salt thereof. In one embodiment, the inhibitor is the corresponding hydrochloride (HCl) salt of the above compound, or other acid addition salt on the piperazine moiety of the above compound. R comprises a CI-C4 alkyl group. [002801 In another embodiment, the 17B-HSD3 inhibitor has the following prodrug structure

OP(O)(OH)

2 H S-N N . / - \ SN HO H

CF

3 [002811 or a pharmaceutically acceptable salt thereof. In one embodiment, the inhibitor is the corresponding hydrochloride (HCI) salt of the above compound, or other acid addition salt on the piperazine moiety of the above compound. [00282] In another embodiment, there are included still further inhibitors of 17B-HSD3, having the structure WO 2012/129673 PCT/CA2012/000316 -97 0 H R2

R

2 RRR 0 wherein R' is alkyl, aralkyl, heterocyclyl, cycloalkyl, -CH 2

CH

2

S(O)

2 -aryl, heterocyclyl,

-CH

2

CH

2 CH(O-acyl)CH 2 -heterocyclyl, or -CH 2

CH

2 NRaRb, wherein Ra and Rb are each independently alkyl, cycloalkyl, acyl, or aralkyl; and each R2 is independently hydrogen or alkyl, or a pharmaceutically acceptable salt or tautomer thereof In another embodiment, the above compound has the structure: O R2 H R R 3 -N / o X, wherein X, is alkyl, R 2 is independently hydrogen or alkyl,

R

3 is hydrogen, alkyl or aralkyl, or a pharmaceutically acceptable salt or tautomer thereof. 1002831 In another embodiment, there are included still further inhibitors of 17B-HSD3, having the structure WO 2012/129673 PCT/CA2012/000316 - 98 00 R2 H R4 R2 N HH wherein,

R

2 is independently hydrogen or alkyl,

R

4 is hydrogen or aralkyl,

R

5 is hydrogen, alkyl or aralkyl, or a pharmaceutically acceptable salt or tautomer thereof. [002841 Inhibition of 170-HSD3 in Intact Cells 100285] After having established the inhibitory activity of the compounds in homogenized HEK-293 cells, the capability of the compounds to exert their inhibitory action in intact cells overexpressing 17p-HSD3 was determined. The results in Table 2 (obtained for intact cells) are indicative of tertiary amine 7a being the best inhibitor. The results in Table 3 indicate that only compounds 10a, 14b, 15b and 15c inhibited over 40% of the activity in homogenized cells (51, 45, 55, 79% inhibition respectively at 0.01 pM. Furthermore, the results in intact cells further illustrated that 15b has the best inhibitory properties. Exhibiting an 84% inhibition at 0.1 pM, compound 15b is a more potent inhibitor in intact cells than 10a, 14b and 15c which exhibited the transformation of 4-dione into T at values of 52, 45 and 47%, respectively. The carbamate-based inhibitors illustrated in Table 4 appear to be less potent inhibitors than either of the series of compounds illustrated in Tables 2 and 3. None of the compounds illustrated in Table 4 provided for an inhibition value exceeding 40% at 0.01 pM in homogenized cells. Moreover, only compounds 17a-i, 22, 23a and 23d exceeded this level of enzyme inhibition at the higher reported concentration of 0.1 ptM. Compound 17a was selected as a representative inhibitor of WO 2012/129673 PCT/CA2012/000316 - 99 this series of carbamate-ADT derivatives in view of iis inhibition of 44% of 17p HSD3 activity in intact cells. 1002861 The IC 50 value of piperazine derivative 15b, the first generation inhibitor 5 and the natural substrate A 4 -dione were determined. Being less potent in both homogenized and intact cell assays, the carbamate derivative 17a was not selected for IC 50 determination. Moreover, the tertiary amine derivative 7a was also not selected as it was shown to be an androgenic compound. From the inhibition curve obtained for intact HEK-293 cells overexpressing 17p-HSD3 (FIG. 12), 15b was found to be an 8-fold better inhibitor than reference compound 5 (IC 50 = 6 and 51 nM, respectively). Compound 15b was also found to be 56-fold better at inhibiting the transformation of labelled A 4 -dione into T (IC 50 = 337 nM) than the unlabelled A 4 dione used itself as an inhibitor. [002871 Proliferative (androgenic) Activity on Shionoqi Cells [00288] In the context of the treatment of androgen-dependent diseases, an important criterion for the development of potential 17p-HSD3 candidates is their non-androgenic character or their ability to not activate the androgen receptor (AR). Accordingly, the agonist (proliferative) activity inhibitors 7a, 15b, 17a, 5 and 3p benzyl-3a-hydroxy-5a-androstan-17-one on androgen-sensitive (ARe) Shionogi cells was evaluated (FIG. 13). In this assay (the basal cell proliferation (control) was set as 100%), the potent androgen dihydrotestosterone (0.1 pM) stimulated cell proliferation to 320% and the antiandrogen (AR antagonist) did not stimulate cell proliferation. Contrary to inhibitor 5, which did not significantly stimulate cell proliferation, the first generation inhibitor CS-213 is fully androgenic, inducing 225% and 396% of basal cell proliferation at 0.1 and 1 pM, respectively. Compound 7a showed a strong proliferative effect (292% and 314% at 0.1 and I pM, respectively). At these two concentrations however, compounds 17a and 15b did not stimulate the proliferation of AR+ cells, thus suggesting no androgenic activity. Sulfonamide 15b is thus a non androgenic inhibitor with strong inhibitory activity (IC 50 = 5 nM) for the WO 2012/129673 PCT/CA2012/000316 - 100 transformation of 4-dione into T in intact transfected HEK-293 cells overexpressing 17P-HSD3. [00289] Additional 17p-HSD3 inhibitors were prepared as illustrated hereinbelow in Schemes 18-21. H H a-c H 0 H HOH NH HO" OI O N- OH H 6 H ADT (1) 2 3 (R = benzyle; isobutyle or H) (R or S, stereochemistry) e N H H R~ HNRH HH N H R 0 6 HN0 0 0 4 8 9 g 0 0 0 H H H N HN H R R HR O A N 0 7 0 0 9 N-substituted azaspirolactones Azaspirolactones Carbamates Scheme 18 WO 2012/129673 PCT/CA2012/000316 - 101 09 S0 0 0 HH H- - H _: HH O HO F H OH H H

-

0 mNH H H / NOHR / 0 3B 0 3CZ 0 3C 0 0A 0 0 H H H H~H H NH OH Ht NHO5H H 0 /0 04 3D 0 3E 0 0 H H H HH H H HN", HN \/ HN- 0HH' 0 H 0*~ H H 4BjI 4 00 0 0 - H HN, H"' H HN, 0 4D 4E Scheme 19 WO 2012/129673 PCT/CA2012/000316 - 102 0 0 0 H H H H A H HH H- HN HN H iH 6' 0 H 0 B0 0 5B 0"j=5 0 0 H HN- A A 0HH HN 5D 0 HE to = 0 oQo H H H HA H~ H H N N N H- 0 OH H 6A y 6 H B6C 0 0 H N A H H 60 0 H N 0 6 0 0 0 H H H H 7B HH7 0 HH- 0 0 H 00 0 o 0 H H H7D 0 H 0 7E Scheme 20 WO 2012/129673 PCT/CA2012/000316 -103 0 0 H 0 H HH 0 N6 N 6 O 00 0/0 9A / 9B 9C 0 0 H H 0 N O0 N-0 H 0 /o 0 9D 9E Scheme 21 [00290] EXPERIMENTAL [002911 Materials, Methods, Synthesis and Characterization [002921 Chemical reagents were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). The usual solvents were obtained from Fisher Scientific (Montreal, QC, Canada) and were used as received. Anhydrous dichloromethane (DCM), dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained from Sigma-Aldrich. Thin-layer chromatography (TLC) and flash-column chromatography were performed on 0.20-mm silica gel 60 F254 plates and with Silicycle R10030B 230-400 mesh silica gel respectively (Qudbec, QC, Canada). Infrared spectra (IR) were recorded using a Perkin Elmer series 1600 FT-IR spectrometer (Norwalk, CT) and the significant bands reported in cm- 1 . Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz for IH and 100.6 MHz for 13C using a Bruker Avance 400 digital spectrometer (Billerica, MA, USA). The chemical shifts (6) were expressed in ppm and referenced to chloroform (7.26 and 77.0 ppm), acetone (2.06 WO 2012/129673 PCT/CA2012/000316 -104 and 29.24 ppm) or methanol (3.31 ppm and 49.0) for 'H and 1C NMR respectively. Low-resolution mass spectra (LRMS) were recorded using a PE Sciex API-150ex apparatus (Foster City, CA, USA) equipped with a turbo ion-spray source and expressed in m/z. High-resolution mass spectra (HRMS) were provided by Pierre Audet at the Departement de Chimie de l'Universit6 Laval (Qu6bec, QC, Canada). The purity of the compounds was determined by high-performance liquid chromatography (HPLC) (Waters Associates Milford, MA, USA) equipped with a UV detector (207 nm) using Luna phenyl hexyl column (75 x 4.6 mm id, 3 piM, serial N 0 : 228048-2, 60 A') or a Nova Pack CI18 reverse-phase column (150 mm x 3.0 mm id, 4 mM, 60A') and a reverse phase column. 1002931 General procedure for synthesis of 30-substituted-3a-hvdroxv androstan-1 7-one derivatives (7a-j) [00294] To a solution of oxirane 650 (50 mg, 0.17 mmol) in anhydrous ethanol (3 mL) was added the appropriate amine (0.5 mmol) and the solution was stirred over a period of 5 h at 60'C. The solvent was subsequently evaporated and the resulting mixture dissolved in dichloromethane (10 mL) followed by the addition of methylisocyanate resin (300 mg, 1.8 mmol/g). The suspension was stirred over a period of 2 h and filtered to provide the corresponding crude amine product. The crude amine was purified by flash chromatography using EtOAc/hexanes (2:8) as the eluent system to provide the desired amines 7a-j. All compounds were characterized by IH NMR and MS analyses. 13 C NMR, HRMS and H-PLC data were also collected for compound 7a. [00295] (3ca,5a)-3{[benzvl(ethyl)aminolmethyl}3-hydroxyandrostan 17-one (7a): (15 mg, 20%); 'H NMR (Acetone-d 6 ): 0.82 (s, 3H), 0.84 (s, 3H), 1.01 (t, J= 7.1 Hz, 3H), 0.80-2.10 (m, 22H), 2.37 (dd,J = 8.7 Hz, J2 = 18.2 Hz, IH), 2.45 (s, 2H), 2.52 (q, J= 7.1 Hz, 2H), 3.74 (s, 2H), 7.23 (t, J= 7.2 Hz, I H), 7.32 (t, J= 7.7 Hz, 2H), 7.39 (d, J= 7.5 Hz, 2H); ' 3 C NMR (CDCl 3 ): 11.3, 12.1, 13.8, 20.2, 21.8, 28.4, 30.8, 31.6, 32.9, 34.0, 35.1, 35.9 (2x), 39.8, 40.8, 47.8, 49.4, 51.4, 54.3, 60.8, WO 2012/129673 PCT/CA2012/000316 - 105 65.3, 70.0, 127.1, 128.4 (2x), 128.6 (2x), 139.5, 221.6; LRMS for C 29

H

44 NO2 [M+H]* 438.2; HRMS calcd for C 29 H4 4

NO

2 [M+H]f 438.3367, found 438.3374; HPLC purity = 99.2% (RT = 7.2 min; 96:4 MeOH/H 2 0; isocratic). [002961 (3a,5a)-3-Ilbenzyl(methyl)aminolmethyll-3 hydroxyandrostan-17-one (7b): (29 mg, 41%); 'H NMR (Acetone-d 6 ): 0.82 (s, 3H), 0.84 (s, 3H), 0.80-2.10 (m, 22), 2.25 (s, 3H), 2.37 (dd, J, = 9.0 Hz, J2 = 18.4 Hz, IH), 2.41 (s, 2H), 3.63 (s, 2H), 7.24 (t, J= 7.2 Hz, IH), 7.32 (t, J= 7.4 Hz, 2H), 7.37 (d, J = 7.2 Hz, 2H); LRMS for C 28 H4 2

NO

2 [M+H]* 424.2. 1002971 (3a,5a)-3-[(diethylamino)methyll-3-hydroxyandrostan-17-one (7c): (40 mg, 65%); 'H NMR (Acetone-d 6 ): 0.81 (s, 3H), 0.84 (s, 3H), 0.99 (t, J= 7.1 Hz, 6H), 0.75-2.08 (m, 22H), 2.37 (s, 2H), 2.30 (dd, J, = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.58 (q, J= 7.1 Hz, 4H); LRMS for C 24

H

42

NO

2 [M+H] 376.2. 100298] (3a,5a)-3-[(dibutylamino)methyll-3-hydroxyandrostan-17-one (7d): (16 mg, 24%); 'H NMR (Acetone-d 6 ): 0.81 (s, 3H), 0.84 (s, 3H), 0.91 (t, J= 7.1 Hz, 6H), 0.80-2.10 (m, 30H), 2.32 (s, 2H), 2.37 (dd, J; = 8.7 Hz, 12 = 18.1 Hz, IH), 2.51 (t, J= 7.4 Hz, 4H); LRMS for C 28

H

5 oNO 2 [M+H]* 432.2. [00299] (3a,5a)-3-hydroxy-3-(piperidin-1-ylmethyl)androstan-17-one (7e): (25 mg, 39%); 'H NMR (Acetone-d6): 0.81 (s, 3H), 0.84 (s, 3H), 0.75-2.09 (m, 28H), 2.18 (s, 2H), 2.37 (dd, J; = 8.6 Hz, J2 = 18.2 Hz, IH), 2.52 (broad s, 4H); LRMS for C 25

H

42

NO

2 [M+H]f 388.2. [003001 (3a,5a)-3-[(4-benzvlpiperidin-1-yl)methyll-3 hydroxyandrostan-17-one (7f): (34 mg, 43%): 'H NMR (Acetone-d 6 ): 0.81 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 27H), 2.20 (s and m, 4H), 2.37 (dd, J, = 8.7 Hz, J2 = 18.2 Hz, IH), 2.53 (d, J= 6.9 Hz, 2H), 2.88 (broad d, J= 11.4 Hz, 2H), 7.18 (d, J= 6.7 Hz, 3H), 7.27 (t, J= 7.4 Hz, 2H); LRMS for C 32

H

4 8

NO

2 [M+H]- 478.1.

WO 2012/129673 PCT/CA2012/000316 -106 1003011 (3ac,5ac)-3-(1,4'-bipiperidin-l'-ylmethyl)-3-hydroxyandrostan 17-one ( 7 g): (28 mg, 44%): 'H NMR (Acetone-d 6 ): 0.81 (s, 3H), 0.84 (s, 3H), 0.75 2.15 (m, 33H), 2.21 (s, 2H), 2.25 (m, 2H), 2.38 (dd, J, = 8.8 Hz, J 2 = 18.2 Hz, IH), 2.47 (t, J= 4.9 Hz, 4H), 2.93 (d, J= 11.5 Hz, 2H); LRMS for C 30

H

51

N

2 0 2 [M+H]* 471.3. 100302] ( 3 a.5a)- 3 -hvdroxy-3-[(4-methylpiperazin-1 yl)methyllandrostan-17-one (7h): (29 mg, 43%): 'H NMR (Acetone-d 6 ): 0.81 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 22H), 2.16 (s, 3H), 2.23 (s, 2H), 2.34 (broad m, 4H), 2.37 (dd, JI = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.58 (broad s, 4H); LRMS for C 25

H

43

N

2 0 2 [M+H]- 403.2. [00303]

(

3 a,5a)-3-hydroxy-3-[(4-phenylpiperazin-1 yv)methyllandrostan-17-one (71): (54 mg, 70%); 'H NMR (Acetone-d 6 ): 0.83 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 21 H), 2.32 (s, 2H), 2.37 (dd, J, = 8.7 Hz, J 2 = 18.2 Hz, I H), 2.75 (m, 4H), 3.18 (m, 4H), 6.77 (t, J= 7.3 Hz, I H), 6.94 (d, J= 8.6 Hz, 2H), 7.21 (d, J= 8.7 Hz, 2H); LRMS for C 30

H

45

N

2 02 [M+H]f 465.0. [003041

(

3 a,5a)- 3 -[(4-benzylpiperazin-1-yI)methyll-3 hydroxyandrostan-17-one (7): (60 mg, 76%): 'H NMR (Acetone-d 6 ): 0.81 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 22H), 2.24 (s, 2H), 2.37 (dd, J = 8.8 Hz, J 2 = 18.3 Hz, 1H), 2.43 (broad m, 4H), 2.61 (s, 4H), 3.47 (s, 2H), 7.23 (m, 111), 7.32 (m, 4H); LRMS for C 31

H

47

N

2 0 2 [M+H]f 478.9. [003051 Synthesis of intermediates 8 and 9 1003061 To a solution of oxirane 650 (1.0 g, 3.3 mmol) in anhydrous ethanol (15 mL) was added either piperazine (573 mg, 6.7 mmol) or trans-2,5 dimethylpiperazine (755 mg, 6.7 mmol). The solution was stirred over a period of 5 h at 60'C. The resulting solution was subsequently poured into water and extracted WO 2012/129673 PCT/CA2012/000316 - 107 three times with EtOAc. The combined organic layers were washed with brine, dried with MgSO 4 , filtered and evaporated under reduced pressure to provide either crude compound 8 or 9. Pure compounds were obtained following flash chromatography using EtOAc/hexanes (1:1) as the eluent system. [00307] ( 3 a,5c)- 3 -hvdroxy-3-(piperazin--ylmethyl)androstan-17-one .8: (750 mg, 61%); 'H NMR (MeOH-d 4 ): 0.84 (s, 3H), 0.89 (s, 3H), 1.01 (t, J= 7.1 Hz, 3H), 0.80-2.15 (m, 20H), 2.25 (s, 2H), 2.45 (dd, J, = 8.6 Hz, J 2 = 19.2 Hz, 1H), 2.58 (broad s, 4H), 2.84 (t, J= 4.8 Hz, 4H). [00308] (3cc,5a)-3-{[(2 ,5 )-2,5-dimethylpiperazin-1-yllmethyl}-3 hydroxyandrostan-17-one (9): (650 mg, 53%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.85 (s, 3H), 0.99 (d, J= 6.0 Hz, 3H), 1.00 (d, J= 6.1 Hz, 3H), 0.75-1.85 (m, 22H), 1.94 (m, 1H), 2.04 (m, 3H), 2.25 (m, 2H), 2.43 (dd, J, = 8.6 Hz, J2 = 18.9 Hz, 1H), 2.54 (t, I H), 2.61 (d, J= 13.9 Hz, IH), 2.90 (m, 3 H); LRMS for C 26

H

45

N

2 0 2 [M+H]f 417.3. 1003091 General Procedure for the synthesis of amines 10a-j and 11a-i [00310] To a solution of compound 8 (25 mg, 0.06 mmol) or compound 9 (30 mg, 0.07 mmol) in anhydrous dichloromethane (5 mL) was added triethylamine (24 mg, 0.24 mmol, 33 iL) and the appropriate benzyl bromide (0.12 mmol). The mixture was stirred overnight and the resulting solution evaporated and purified by flash chromatography using EtOAc/hexanes (3:7) as the eluant system to provide benzylamines 10a-j and lla-j. All compounds were characterized by 'H NMR and MS analyses. "C NMR, HRMS and HPLC data were also collected for compounds 10a and 11a. [003111 (3a,5a)-3-hydroxy-3-({4-[3-(trifluoromethyl)benzvllpiperazin 1-yllmethyl)androstan-17-one (10a): (31 mg, 97%); 'H NMR (CDCl 3 ): 0.76 (s, 3H), 0.86 (s, 3H), 0.75-2.10 (m, 22H), 2.27 (s, 2H), 2.44 (broad m, 5H), 2.66 (broad s, WO 2012/129673 PCT/CA2012/000316 - 108 4H), 3.54 (s, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.50 (broad d, J = 7.5 Hz, 2H), 7.58 (s, IH); "C NMR (CDC] 3 ): 11.2, 13.8, 20.2, 21.8, 28.3, 30.8, 31.5, 32.7, 33.8, 35.1, 35.8, 35.9, 39.6, 40.7, 47.8, 51.4, 54.2, 55.7 (4x), 62.3, 69.0, 70.1, 124.0 (q, JC-CC-F= 3.6 Hz), 124.2 (q, Jc-F = 272 Hz), 125.6 (q, JC-C-C-F = 3.8 Hz), 128.7, 130.6 (q, JC-C-F = 32 Hz), 132.4, 139.2, 221.5; LRMS for C 32 1- 46

F

3

N

2 0 2 [M+H]+ 547.3; HRMS called for C 32

H

46

F

3

N

2 0 2 [M+H]f 547.3506, found 547.3510; HPLC purity: 97.2% (RT = 5.5 min; 96:4 MeOH/H 2 0; isocratic). 100312] (3a,5a)-3-hydroxy-3-({4-[2-(trifluoromethyl)benzvllpiperazin 1-yllmethyl)androstan-17-one (10b): (33 mg, 45%); 'H NMR (CDC1 3 ): 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.27 (s, 2H), 2.43 (dd, J = 8.7 Hz, J2 = 19.2 Hz, I H), 2.50 (broad s, 4H), 2.66 (broad, s, 4H), 3.65 (s, 2H), 7.33 (t, J= 7.7 Hz, 1H), 7.51 (d, J= 6.7 Hz, 1 H), 7.62 (d, J= 7.8 Hz, I H), 7.77 (d, J= 7.7 Hz, 1H); LRMS for

C

32

H

46

F

3

N

2 0 2 [M+H]* 547.1. 1003131 (3a,5a)-3-hydroxy-3-({4-[4-(trifluoromethyl)benzyllpiperazin 1-yI}methyl)androstan-17-one (10c): (32 mg, 97%); 'H NMR (CDC1 3 ): 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.27 (s, 2H), 2.45 (broad m, 5H), 2.65 (broad s, 4H), 3.54 (s, 2H); 7.44 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H); LRMS for

C

32

H

46

F

3

N

2 0 2 [M+H]± 547.2. [003141 (3ac,5a)-3-hvdroxy-3-[(4-{3 [(trifluoromethyl)sulfanyll benzvl Ipiperazin-1-yA)methyI androstan-17-one (10d): (25 mg, 71%); 1 H NMR (CDC 3 ): 0.76 (s, 3H), 0.86 (s, 3H), 0.78-2.10 (m, 22H), 2.27 (s, 2H), 2.45 (broad n, 5H), 2.65 (broad s, 4H), 3.52 (s, 2H), 7.37 (t, J= 7.6 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.62 (s, 1H); LRMS for

C

32 H4 6

F

3

N

2 0 2 S [M+H]f 579.3. 100315] (3a,5a)-3-1[ 4

-(

3 -chlorobenzyl)piperazin-1-yllmethyl}-3 hydroxyandrostan-17-one (10e): (31 mg, 94%); 'H NMR (CDCl 3 ): 0.76 (s, 3H), WO 2012/129673 PCT/CA2012/000316 -109 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.27 (s, 2H), 2.43 (broad m, 5H), 2.65 (broad s, 4H), 3.46 (s, 2H); 7.19 (m, IH), 7.23 (s, 2H), 7.33 (s, 1H); LRMS for C 31

H

4 6 ClN 2 0 2 [M+H]+ 513.3. [003161 (3a,5a)-3-hydroxy-3-( 14-(3-methoxybenzvl)piperazin-1 yllmethyllandrostan-17-one (10f): (21 mg, 64%); 'H NMR (CDC 3 ): 0.76 (s, 3H), 0.85 (s, 3H), 0.78-2.10 (m, 22H), 2.26 (s, 2H), 2.43 (broad m, 5H), 2.65 (broad s, 4H), 3.48 (s, 2H), 3.81 (s, 3H), 6.80 (d, J= 7.5 Hz, 1 H), 6.88 (s, 1H), 6.89 (d, J= 7.8 Hz, 1H), 7.23 (t, J= 8.0 Hz, IH); LRMS for C32H49N203 [M+H] 509.3. [003171 (3t,5c)-3-hydroxy-3-f 14-(pyridin-3-ylmethyl)piperazin-1 yllmethyllandrostan-17-one (10g): (20 mg, 65%); 'H NMR (CDC 3 ): 0.76 (s, 3H), 0.85 (s, 3H), 0.78-2.10 (m, 22H), 2.27 (s, 2H), 2.43 (broad m, 5H), 2.65 (broad s, 4H), 3.51 (s, 2 H), 7.24 (d, J= 4.9 Hz, I H), 7.65 (d, J= 7.8 Hz, I H), 8.51 (dd, J, = 1.3 Hz, J2= 4.8 Hz, IH), 8.54 (d, J= 1.4 Hz, 1H); LRMS for C 3 0

H

46

N

3 0 2 [M+H] 480.2. [00318] (3a,5a)-3-({4-[2,5-bis(trifluoromethyl)benzyllpniperazin-1 yllmethyl)-3-hydroxyandrostan-17-one (10h): (27 mg, 73%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.78-2.10 (m, 22H), 2.29 (s, 2H), 2.43 (dd, J, = 8.7 Hz, J2= 19.3 Hz, 1H), 2.51 (broad s, 4H), 2.68 (broad s, 4H), 3.69 (s, 2H), 7.59 (d, J = 8.1 Hz, I H), 7.75 (d, J = 8.2 Hz, I H), 8.12 (s, I H); LRMS for C 33 H4sF 6

N

2

O

2 [M+H]* 615.5. [00319] ( 3 a, 5 a)- 3 -({4-12,4-bis(trifluoromethyl)benzyll piperazin-1 yllmethvl)-3-hydroxyandrostan-17-one (10i): (27 mg, 73%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.83-2.10 (m, 22H), 2.28 (s, 2H), 2.43 (dd, J = 8.5 Hz, J2 = 19.1 Hz, 1 H), 2.51 (broad s, 4H), 2.67 (broad s, 4H), 3.70 (s, 2H), 7.77 (d, J= 8.3 Hz, I H), 7.88 (s, I H), 7.98 (d, J= 8.2 Hz, I H); LRMS for C 33

H

4 5

F

6

N

2 0 2 [M+H]+ 615.2.

WO 2012/129673 PCT/CA2012/000316 - 110 [003201 (3c,5a)-3-({4-13,5-bis(trifluoromethyl)benzyll piperazin-1 yllmethyl)-3-hydroxyandrostan-17-one (10j): (29 mg, 78%); H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.28 (s, 2H), 2.43 (broad m, 5H), 2.67 (broad s, 4H), 3.59 (s, 2H), 7.77 (s, IH), 7.79 (s, 2H); LRMS for C 33

H

45

F

6

N

2 0 2 [M+H]f 615.5. [003211 (3a,5a)-3-({{2,5 )-2,5-dimethyl-4-13 (trifluoromethyl)benzvllpiperazin-1-yl}methyl)-3-hydroxyandrostan-17-one (1.a): (25 mg, 71%); lH NMR (CDC 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d, J = 6.2 Hz, 3H), 1.10 (d, J= 6.0 Hz, 3H), 0.80-2.10 (m, 23H), 2.32 (m, 6H), 2.91 (d, J = 11.1 Hz, I H), 3.11 (d, J= 13.7 Hz, I H), 3.22 (d, J= 16.1 Hz, IH), 4.07 (d, J= 13.7 Hz, IH), 7.43 (m, IH), 7.51 (d, J= 7.8 Hz, 2H), 7.58 (s, 1H); 1 3 C NMR (CDC1 3 ): 11.3, 13.8 (3x), 20.2, 21.8, 28.3, 30.8, 31.6, 32.7, 33.9, 35.1, 35.9 (2x), 39.4, 40.8, 47.8, 51.4, 54.3, 55.8, 56.3, 57.5, 63.8, 69.4, 123.7 (q, JC-C-C-F = 3.6 Hz), 125.0 (q, J-P = 273 Hz), 125.4 (q, JC-C-C-F = 3.6 Hz), 128.6, 130.5 (q, JC-CF- = 32 Hz), 132.1, 140.3, 221.6; LRMS for C 34

H

5 oF 3

N

2 0 2 [M+H]± 575.2; HRMS calcd for C 34

H

5 oF 3

N

2 0 2 [M+H]f 575.3819, found 575.3826; HPLC purity: 99.0% (RT = 6.6 min; 75:25 to 5:95 MeOH/H 2 0 isocratic gradient). [00322] ( 3 a,5a)-3-({(2,5 )-2,5-dimethyl-4-[2 (trifluoromethyl)benzvllpiperazin-1-yllmethv)-3-hydroxyandrostan-17-one (_1b): (17 mg, 49%); 1 H NMR (CDC1 3 ): 0.78 (s, 3H), 0.86 (s, 3H), 0.93 (d, J= 6.2 Hz, 3H), 1.05 (d, J= 6.0 Hz, 3H), 0.75-2.10 (m, 23H), 2.33-2.60 (m, 6H), 2.93 (d, J= 11.1 Hz, IH), 3.23 (broad s, IH), 3.31 (d, J= 15.1 Hz, IH), 4.07 (d, J= 14.8 Hz, 1 H), 7.31 (t, J= 7.7 Hz, 1H), 7.51 (t, J= 7.4 Hz, I H), 7.61 (d, J= 7.9 Hz, IH), 7.87 (d, J= 7.8 Hz, IH); LRMS calcd for C 34

H

5 oF 3

N

2 0 2 [M+H]* 575.3. [00323] (3a,5a)-3-({(2 ,5 )-2,5-dimethyl-4-[4 (trifluoromethyl)benzyllpiperazin-1-yl methyl)-3-hydroxyandrostan-17-one (1_c1: (22 mg, 63%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d, J = 6.2 WO 2012/129673 PCT/CA2012/000316 -111 Hz, 3H), 1.10 (d, J= 6.0 Hz, 3H), 0.75-2.15 (m, 24H), 2.30-2.60 (m, 6H), 2.91 (d, J 11.5 Hz, I H), 3.11 (d, J= 13.6 Hz, 1H), 3.20 (broad s, IH), 4.07 (d, IH, J= 13.9 Hz, 1H), 7.37 (d, J = 7.7 Hz, IH), 7.43 (d, J = 8.0 Hz, IH), 7.57 (d, J = 8.1 Hz, IH); LRMS for C 34

H

5 oF 3

N

2 0 2 [M+H]* 575.2. [00324] (3a,5a)-3-{[(2 ,5E)-2,5-dimethyl-4-{3 [(trifluoromethyl)sulfanyll benzyllpiperazin-1-yllmethyl}-3-hydroxyandrostan 17-one (ld): (15 mg, 41%); 'H NMR (CDC1 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d, J = 6.2 Hz, 3H), 1.10 (d, J= 6.0 Hz, 3H), 0.75-2.10 (m, 23H), 2.30-2.60 (m, 6H), 2.91 (d, J= 10.5 Hz, IH), 3.10 (d, J= 13.2 Hz, IH), 3.21 (d, J= 16.2 Hz, 1H), 4.04 (d, J= 13.1 Hz, IH), 7.36 (t, J= 7.7 Hz, I H), 7.43 (d, J= 7.7 Hz, 1H), 7.53 (d, J= 8.5 Hz, I H), 7.62 (s, I H); LRMS for C 34

H

5 oF 3

N

2 0 2 S [M+H]f 607.2, [00325] (3a,5a)-3-{[(2 ,5 )-4-(3-chlorobenzv)-2,5-dimethvpiperazin 1-yllmethyl}-3-hydroxyandrostan-17-one (11e): (12 mg, 37%); 1H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.93 (d, J= 6.2 Hz, 3H), 1.09 (d, J= 6.0 Hz, 3H), 0.75-2.10 (m, 23H), 2.30-2.62 (m, 6H), 2.89 (d, J = 11.2 Hz, 1H), 3.03 (d, J = 13.4 Hz, 1H), 3.22 (broad s, 1H), 4.00 (d, J= 13.3 Hz, IH), 7.18 (t, J= 7.2 Hz, IH), 7.23 (s, 2H), 7.33 (s, 1H); LRMS for C 3 3

H

50 35 ClN 2 0 2 [M+H]f 541.3. [00326] ( 3 a,5a)-3-hvdroxy-3-{[(2,5)-4-(3-methoxybenzl)-2,5 dimethylpiperazin-1-yilmethyllandrostan-17-one (I1f): (14 mg, 44%); 1H NMR (CDC1 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d, J= 6.0 Hz, 3H), 1.11 (d, J= 5.3 Hz, 3H), 0.75-2.10 (m, 24H), 2.30-2.80 (m, 6H), 2.89 (d, J= 10.2 Hz, IH), 3.07 (d J= 13.2 Hz, IH), 3.81 (s, 3H), 4.01 (d, J= 12.8 Hz, IH), 6.79 (m, IH), 6.89 (m, 2H), 7.22 (m, I H); LRMS for C 34

H

53

N

2 0 3 [M+H]f 537.4. [003271 ( 3 a,5a)- 3 -{[(2 ,55)-2,5-dimethyl-4-(pyridin-3 ylmethyl)piperazin-1-yllmethyll-3-hydroxyandrostan-17-one (11g): (10 mg, 33%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d,J= 6.0 Hz, 3H), 1.12 (d, WO 2012/129673 PCT/CA2012/000316 - 112 J= 5.4 Hz, 3H), 0.75-2.10 (m, 22H), 2.30-2.68 (m, 7H), 2.90 (d, J= 11.0 Hz, IH), 3.10 (m, 2H), 4.02 (d, J= 13.2 Hz, 1H), 7.25 (s, 1H), 7.64 (m, 1H), 8.52 (m, 2H); LRMS for C 32

H

5 oN 3 0 2 [M+H]* 508.3. 1003281 (3a,5a)-3-({(2 ,5 )-4-[2,5-bis(trifluoromethyl)benzvll-2,5 dimethylpiperazin-1-yl methyl)-3-hydroxyandrostan-17-one (11h): (46 mg, 98%); 'H NMR (CDCl 3 ): 0.78 (s, 3H), 0.86 (s, 3H), 0.95 (d, J= 6.1 Hz, 3H), 1.03 (d, J= 6.0 Hz, 3H), 0.75-2.10 (m, 23H), 2.35-2.60 (m, 6H), 2.95 (d, J = 11.6 Hz, 1H), 3.18 (broad s, I H), 3.40 (d, J= 15.8 Hz, IH), 4.06 (d, J= 15.8 Hz, 1H), 7.58 (d, J= 8.1 Hz, 1H), 7.74 (d, J = 8.2 Hz, IH), 8.22 (s, IH); LRMS for C 35

H

49

F

6

N

2 0 2 [M+H]* 643.3. 1003291 (3a,5a)-3-({(2 ,5 )-4-[2,4-bis(trifluoromethyl)benzvll-2,5 dimethylpiperazin-1-yllmethyl)-3-hydroxyandrostan-17-one (11i): (46 mg, 98%); 'H NMR (CDC 3 ): 0.78 (s, 3H), 0.86 (s, 3H), 0.94 (d, J= 6.1 Hz, 3H), 1.03 (d, J= 6.1 Hz, 3H), 0.75-2.10 (m, 23H), 2.32-2.60 (m, 6H), 2.94 (d, J = 11.0 Hz, 1H), 3.13 (broad s, IH), 3.39 (d, J= 15.8 Hz, IH), 4.08 (d, J= 15.4 Hz, IH), 7.76 (d, J= 8.2 Hz, IH), 7.86 (s, IH), 8.08 (d, J = 8.2 Hz, 1H); LRMS for C 35

H

49

F

6

N

2 0 2 [M+H]* 643.3. [003301 (3a,5a)-3-f(2 ,5 )-4-[3,5-bis(trifluoromethyl)benzvll-2,5 dimethylpiperazin-1-yl methyl)-3-hydroxyandrostan-17-one (11i): (20 mg, 43%); iH NMR (CDCI): 0.78 (s, 3H), 0.86 (s, 3H), 0.94 (d, J= 6.1 Hz, 3H), 1.09 (d, J= 6.1 Hz, 3H), 0.75-2.12 (m, 24H), 2.35-2.57 (m, 6H), 2.93 (d, J= 11.4 Hz, IH), 3.18 (d, J = 11.4 Hz, 1H), 4.09 (d, J= 13.3 Hz, IH), 7.76 (s, IH), 7.80 (s, 2H); LRMS for

C

35 H4 9

F

6

N

2 0 2 [M+H]+ 643.4. (00331] General Procedure for Synthesis ofAmides 12a-e and 13a-d WO 2012/129673 PCT/CA2012/000316 -113 100332] To a solution of compound 8 (40 mg, 0.1 mmol) or compound 9 (25 mg, 0.06 mmol) in anhydrous DCM (3 mL) was added triethylamine (4.0 eq) and the appropriate acyl chloride (2.0 eq). The solution was then stirred over a period of 3 h at room temperature. The resulting solution was concentrated and purified by flash chromatography using EtOAc/hexanes (7:3 to 9:1) as to eluant system to provide the corresponding amides 12a-e and 13a-d. All compounds were characterized by IH NMR and MS analyses. 1C NMR, HRMS and HPLC data were also collected for compounds 12a, 12c and 13a. 100333] (3a.,5a)-3-hydroxy-3-{14-(phenylcarbonyl)piperazin-1 yllmethyllandrostan-17-one (12a): (33 mg, 64%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (in, 22H), 2.31 (s, 2H), 2.43 (dd, J, = 8.7 Hz, J 2 = 19.2 Hz, 1H), 2.57 and 2.70 (broad 2s, 4H), 3.43 (broad s, 2H), 3.79 (broad s, 2H), 7.40 (m, 5H); 13 C NMR (CDCI 3 ) 5 11.2, 13.8, 20.2, 21.7, 28.3, 30.8, 31.5, 32.4, 33.6, 35.0, 35.8, 35.9, 39.3, 40.6, 47.8, 51.4, 54.1, 55.4 (2x), 55.7 (2x), 69.1, 70.5, 127.0 (2x), 128.5 (2x), 129.8, 135.5, 170.3, 221.5; LRMS for C 31

H

45

N

2 0 3 [M+H] 493.0; HRMS calcd for C 31

H

45

N

2 0 3

[M+H]

t 493.3425, found 493.3433; HPLC purity: 94.8% (RT = 10.2 min; 75:25 to 5:95 MeOH/H 2 O isocratic). [003341 ( 3 a,5a)-3-{[4-(cyclohexylcarbonyl)piperazin-1-yllmethyl}-3 hydroxyandrostan-17-one (12b): (30 mg, 56%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (m, 21H), 2.29 (s, 2H), 2.43 (in, 2H), 2.60 (m, 4H), 2.92 (broad s, I H), 3.49 (broad s, 2H), 3.61 (broad s, 2H); LRMS for C 31

H

51

N

2 0 3 [M+H]* 499.3. 100335] (3a,5a)-3-hvdroxy-3-[(4-{[2 (trifluoromethyl)phenyllcarbonyl}-piperazin-1-yl)methyllandrostan-17-one (12c: (30 mg, 54%); 1H NMR (CDCl 3 ): 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (in, 22H), 2.30 (s, 2H), 2.43 (dd, J = 8.7 Hz, J 2 = 19.1 Hz, 1H), 2.53 (m, 2H), 2.70 (broad s, 2H), 3.18 (t, J= 5.0 Hz, 2H), 3.82 (broad m, 2H), 7.32 (d, J= 7.4 Hz, 11-), 7.52 (t, J WO 2012/129673 PCT/CA2012/000316 -114 7.6 Hz, 1 H), 7.60 (t, J= 7.5 Hz, 1H), 7.71 (d, J= 7.8 Hz, I H); 13C NMR (CDCl 3 ): 11.2, 13.8, 20.2, 21.7, 28.3, 30.8, 31.5, 32.3, 33.6, 35.1, 35.8, 35.9, 39.3, 40.6, 41.9, 47.3, 47.8, 51.4, 54.1, 55.1, 55.2, 69.0, 70.6, 123.6 (q, Jc-F = 274 Hz), 126.6 (q, Jc-c c. F = 4.4 Hz), 126.7 (q, JC-C-F = 32 Hz), 127.2, 129.2, 132.2, 134.7, 167.3, 221.4; LRMS for C 32

H

44

F

3

N

2 0 3 [M+H]f 561.1; HRMS called for C 32

H

44

F

3

N

2 0 3 [M+H]* 561.3299, found 561.3304; HPLC purity: 96.7% (RT = 10.7 min; 75:25 to 5:95 MeOH/H 2 0 isocratic). [00336] (3a,5a)-3-hydroxy-3-[(4-{ [3 (trifluoromethyl)phenyllcarbonyllpiperazin-1-yI)methyllandrostan-17-one (12d): (42 mg, 69%); 'H NMR (CDC1 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (m, 22H), 2.32 (s, 2H), 2.43 (dd, J = 8.7 Hz, J2 = 19.3 Hz, I H), 2.59 (broad s, 2H), 2.72 (broad s, 2H), 3.41 (broad s, 2H), 3.80 (broad s, 2H), 7.55 (m, 2H), 7.67 (s, 2H); LRMS for

C

32 4 4

F

3

N

2 0 3 [M+H]* 561.4. [003371 (3a,5a)-3-hydrox-3-[(4-14 (trifluoromethyl)phenyllcarbonyllpiperazin- 1-yI)methyll androstan-17-one (12e): (44 mg, 72%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.32 (s, 2H), 2.43 (dd, J, = 8.7 Hz, J2 = 19.3 Hz, IH), 2.57 (broad s, 2H), 2.73 (broad s, 2H), 3.39 (broad s, 2H), 3.80 (broad s, 2H), 7.51 (d, J= 8.0 Hz, 2H), 7.68 (d, J= 8.1 Hz, 2H); LRMS for C 32 H4 4

F

3

N

2 0 3 [M+HJr 561.2. [003381 ( 3 a,5a)- 3

-{[(

2 ,5)-2,5-dimethyl-4-(phenylcarbonyl)piperazin 1-yllmethyl}-3-hydroxyandrostan-17-one (13a): (25 mg, 31%); 'H NMR (CDC1 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 1.27 (m, 3H), 1.41 (d, J = 6.8 Hz, 3H), 0.75-2.00 (m, 23H), 2.05-2.17 (m, 2H), 2.34-2.47 (m, 3H), 2.90 (broad s, IH), 3.06 (d, J= 8.8 Hz, 1H), 3.50 (broad s, 1H), 7.30-7.42 (m, 5H); 1 3 C NMR (CDCl 3 ) 6 8.6, 11.2, 13.8 (2x), 20.2, 21.7, 28.3, 30.8, 31.5, 32.5, 33.8, 35.0, 35.8, 35.9, 39.4, 40.7, 47.8, 51.4, 51.7, 54.0, 54.2, 55.1 (2x), 65.8, 70.9, 126.5, 128.5 (2x), 129.3 (2x), 136.4, 171.2, 221.5; LRMS for C 33 H4 9

N

2 0 3 [M+H]f 521.4; HRMS calcd for C 33 I-4 9

N

2 0 3 [M+H]f 521.3738, WO 2012/129673 PCT/CA2012/000316 - 115 found 521.3745; HPLC purity: 96.4% (RT = 12.1 min; 75:25 to 5:95 MeOH/H 2 0 isocratic). 1003391 (3a,5a)-3-{[(2 ,5 )-4-(cyclohexylcarbonyl)-2,5 dimethylpiperazin-1-yllmethyll-3-hydroxyandrostan-17-one (13b): (14 mg, 44%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 1.27 (m, 6H), 0.80-2.00 (m, 22H), 2.02 2.18 (m, 2H), 2.34-2.47 (m, 3H), 2.90-3.15 (broad m, 3H), 3.46 and 3.60 (2d, J= 11.8 Hz, IH), 4.02, 4.28 and 4.70 (3m, 1H); LRMS for C 33

H

5 5

N

2 0 3 [M+H]* 527.3. 1003401 (3a,5a)-3-{[(24,5 )-2,5-dimethyl-4-{[3 (trifluoromethyl)phenyllcarbonyl}piperazin-1-ylmethyl}-3-hydroxyandrostan 17-one (13c): (27 mg, 77%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 1.26 (m, 3H), 1.43 (d, J= 6.4 Hz, 3H), 0.78-2.00 (m, 22H), 2.02-2.20 (m, 2H), 2.35-2.47 (m, 3H), 2.95 (broad s, 2H), 3.07 (d, J= 10.4 Hz, 1H), 3.55 (broad s, I H), 7.55 (d, J= 4.5 Hz, 2H), 7.61 (s, 1H), 7.68 (broad s, 1H); LRMS for C 34 H4 8

F

3

N

2

O

3

[M+H]

t 589.3. [003411 (3a,5a)-3-{[(2 ,5 )-2,5-dimethyl-4-{[4 (trifluoromethyl)phenyllcarbonyll piperazin-1-yllmethyll-3-hydroxyandrostan 17-one (13d): (31 mg, 89%); 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.86 (s, 3H), 1.27 (m, 3H), 1.42 (m, 3H), 0.80-1.98 (m, 22H), 2.03-2.09 (m, 2H), 2.35-2.47 (m, 3H), 2.95 (broad s, 2H), 3.05 (broad s, I H), 3.58 (broad s, 1H), 7.46 (d, J= 7.9 Hz, 2H), 7.68 (d, J= 8.0 Hz, 2H); LRMS for C 34

H

4 8

F

3

N

2 0 3 [M+H]+ 589.2. 100342] General Procedure for Synthesis of Sulfonamides 14a-c and 15a-c [00343] To a solution of compound 8 (30 mg, 0.08 mmol) or compound 9 (30 mg, 0.07 mmol) in anhydrous dichloromethane (3 mL) was added triethylamine (33 mL, 24 mg, 0.24 mmol) and the appropriate sulfonyl chloride (0.12 mmol). The solution was then stirred over a period of 3 h at room temperature. The resulting WO 2012/129673 PCT/CA2012/000316 - 116 solution was concentrated and purified by flash chromatography using EtOAc/hexanes (3:7 to 1:1) as to eluant system to provide the corresponding sulfonamides 14a-c and 15a-c. All compounds were characterized by 1 H NMR and MS analyses. 1C NMR, HRMS and HPLC data were also collected for compounds 14a, 15b and 15c. [00344] (3a,5a)-3-hvdroxy-3-[(4-{[3 (trifluoromethyl)phenyllsulfonyllpiperazin-1-yl)methyllandrostan-17-one (14a): (29 mg, 63%); 'H NMR (CDC 3 ): 0.74 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 21 H), 2.28 (s, 2H), 2.43 (dd, J, = 8.7 Hz, J 2 = 19.3 Hz, lH), 2.50 (s, lH), 2.72 (t, J= 4.7 Hz, 4H), 3.05 (broad s, 4H), 7.73 (t, J= 7.8 Hz, IH), 7.90 (d, J= 7.8 Hz, 1H), 7.95 (d, J= 7.9 Hz, lH), 8.01 (s, lIH); 1 3 C NMR (CDC 3 ): 11.2, 13.8, 20.2, 21.7, 28.2, 30.7, 31.5, 32.2, 33.6, 35.0, 35.8, 35.9, 39.1, 40.5, 46.3 (2x), 47.8, 51.4, 54.1, 54.7 (2x), 68.8, 70.6, 123.2 (q, JC-F = 273 Hz), 124.7 (q, JC-C-CF = 3.7 Hz), 129.6, 130.0, 130.9, 131.9 (q, JC-C-F = 33 Hz), 137.0, 221.4; LRMS for C 3 1H 43

F

3

N

2 0 4 SNa [M+Na]+ 619.5; HRMS calcd for C 3 1H 44

F

3 N20 4 S [M+HJf 597.2968, found 597.2974; HPLC purity: 98.4 % (RT = 15.4 min; 70:30 to 5:95 MeOH/H 2 0 isocratic). 1003451 (3a,5a)-3-hvdrox-3-[(4-fl2 (trifluoromethyl)phenyllsulfonyll piperazin-1-yl)methyllandrostan-17-one (14b): (33 mg, 72%); 'H NMR (CDC 3 ): 0.75 (s, 3H), 0.85 (s, 3H), 0.75-2.12 (m, 21 H), 2.29 (s, 2H), 2.43 (dd, J = 8.6 Hz, J 2 = 19.2 Hz, IH), 2.69 (in, 5H), 3.26 (broad s, 4H), 7.72 (in, 2H), 7.92 (d, J = 9.1 Hz, IH), 8.10 (d, J = 9.1 Hz, IH); LRMS for

C

3 1H 43

F

3

N

2 0 4 SNa [M+Na]+ 619.2. [003461 (3a,5a)-3-hydroxy-3-[(4-{ [4-(trifluoromethyl)phenyllsulfonyl piperazin-1-yl)methyllandrostan-17-one (14c): (26 mg, 56%); 'H NMR (CDCl 3 ): 0.74 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (in, 23H), 2.27 (s, 2H), 2.43 (dd, J, = 8.6 Hz, J 2 = 19.2 Hz, I H), 2.50 (s, IH), 2.72 (broad t, J= 4.7 Hz, 4H), 3.06 (broad s, 4H), 7.83 (d, WO 2012/129673 PCT/CA2012/000316 -117 J = 8.4 Hz, 1H), 7.89 (t, J = 8.3 Hz, IH); LRMS for C 31

H

43

F

3

N

2 0 4 SNa [M+Na]* 619.3. [00347] (3a,5a)-3-{[(2 ,5 )-2,5-dimethyl-4-{[3 (trifluoromethyl)phenyllsulfonyllpiperazin-1-yllmethyll-3-hydroxyandrostan 17-one (15a): (30 mg, 72%); 'H NMR (CDCl 3 ): 0.75 (s, 3H), 0.85 (s, 3H), 1.00 (d, J = 6.4 Hz, 3H), 1.14 (d, J= 6.8 Hz, 3H), 0.75-2.18 (m, 22H), 2.30-2.46 (m, 3H), 2.69 (s, I H), 2.95 (m, I H), 3.07 (dd, J; = 3.5 Hz, J 2 = 11.9 Hz, 1 H), 3.39 (s, 2H), 4.10 (m, I H), 7.66 (t, J= 7.8 Hz, I H), 7.82 (d, J= 7.9 Hz, I H), 7.99 (d, J= 7.8 Hz, I H), 8.06 (s, 1H); LRMS for C 33

H

47

F

3

N

2 0 4 SNa [M+Na]* 647.4. [00348] (3a,5a)-3-{[(2.,5 )-2,5-dimethyl-4-{[2 (trifluoromethyl)phenyllsulfonyl}piperazin-1-yllmethyll-3-hydroxyandrostan 17-one (15b): (29 mg, 63%); 'H NMR (CDCl 3 ): 0.75 (s, 3H), 0.85 (s, 3H), 0.89 (m, 3H), 1.19 (m, 3H), 0.75-1.98 (m, 19H), 2.00-2.17 (m, 2H), 2.33 (tapp, J= 13.2 Hz, 2H), 2.43 (dd, J, = 8.8 Hz, J2 = 19.2 Hz, I H), 2.82 (s, I H), 2.90 (m, I H), 3.09 (d, J= 11.6 Hz, I H), 3.35 (m, 2H), 3.52 (d, J= 13.0 Hz, 1H), 4.05 (broad s, I H), 7.69 (d, J = 4.5 Hz, 2H), 7.89 (d, J= 9.1 Hz, 1H), 8.18 (d, J= 9.1 Hz, IH); 1 3 C NMR (CDCl 3 ): 8.6, 11.2, 13.8, 15.6, 20.2, 21.7, 28.3, 30.7, 31.5, 32.4, 33.8, 35.0, 35.8, 35.9, 39.3, 40.7, 46.0, 47.8, 49.5, 51.4, 52.4, 54.2, 54.7, 65.7, 70.9, 122.6 (q, JC-F = 274 Hz), 127.5 (q, JC-C-F = 33 Hz), 128.5 (q, Jc-c-c-r = 6.4 Hz), 131.9, 132.1, 132.5, 139.3, 221.5; LRMS for C 33

H

47

F

3

N

2 0 4 SNa [M+Na]f 647.2; HRMS calcd for

C

33 Hs 8

F

3

N

2 0 4 S [M+H]* 625.3281, found 625.3291; HPLC purity: 98.9% (RT = 16.1 min; 70:30 to 5:95 MeOH/H 2 0 isocratic gradient). [00349] (3ac,5a)-3-{[(2 ,5E)-2,5-dimethyl-4-{[12-, trifluoromethyl)phenyllsulfonyllpiperazin-1-yll methyll-3-hydroxyandrostan-17 one (15c): (35 mg, 70%); 'H NMR (CDCl 3 ): 0.75 (s, 3H), 0.85 (s, 3H), 1.01 (d, J= 5.9 Hz, 3H), 1.14 (d, J= 6.7 Hz, 3H), 0.75-1.98 (m, 20H), 2.00-2.18 (m, 2H), 2.30 2.45 (m, 3H), 2.68 (s, 1H), 2.95 (d, J= 6.6 Hz, I H), 3.07 (dd, J; = 3.6 Hz, J2 = 11.8 WO 2012/129673 PCT/CA2012/000316 - 118 Hz, 1H), 3.40 (s, 2H), 4.05 (broad s, IH), 7.77 (d, J= 8.3 Hz, 2H), 7.93 (d, J= 8.3 Hz, 2H); 13 C NMR (CDC 3 ): 9.1, 11.2, 13.8, 14.9, 20.2, 21.7, 28.3, 30.7, 31.5, 32.4, 33.7, 35.0, 35.8, 35.9, 39.3, 40.7, 46.1, 47.8, 49.9, 51.4, 53.0, 54.2, 54.6, 65.6, 71.0, 123.2 (q, JC-F = 273 Hz), 126.1, 126.2 (q, JC--c-- = 3.6 Hz), 127.4 (2x), 134.1 (q, Jc-c F = 33 Hz), 144.2, 221.5; LRMS for C 33

H

47

F

3

N

2 0 4 SNa [M+Na]+ 647.4; HRMS called for C 33 H4 8

F

3

N

2 0 4 S [M+H]- 625.3281, found 625.3287; HPLC purity: 98.7% (RT = 17.0 min; 70:30 to 5:95 MeOH/H 2 0 isocratic). [00350] General Procedure for Synthesis of 3-Carbamate-androsterone derivatives 17a-1 7i [00351] To a solution of oxirane 650 (75 mg, 0.25 mmol) in anhydrous ethanol (5 mL) was added the appropriate primary amine (0.75 mmol). The solution was subsequently stirred overnight at 70'C. The resulting solution was subsequently concentrated and purified by flash chromatography using EtOAc/hexanes (7:3 to 9:1) as the eluent system to provide the corresponding secondary amine product 16 in good yields (70-90%). [00352] To a solution of compound 16 (0.22 mmol) in anhydrous DCM (7 mL), at 0 0 C and under an argon atmosphere, was added diisopropylamine (0.66 mmol) and triphosgene (0.11 mmol). The solution was subsequently stirred overnight at room temperature. The reaction mixture was then poured into water, extracted with DCM, filtered using a phase separator (Biotage, Uppsala, Sweden) and concentrated. The crude compounds were purified by flash chromatography using EtOAc/hexanes (1:9 to 3:7) as the eluant system to provide the corresponding carbamates 17a-i. All compounds were characterized by IH NMR and MS analyses. 13 C NMR, HRMS and HPLC data were also collected for compound 17a. [00353] (3R, 5S, 8R, 9S, 10S, 13S, 14S)-3'-benzyl-10,13 dimethyltetradecahydro-2'H spiro[cyclopentaltl phenanthrene-3,5'- WO 2012/129673 PCT/CA2012/000316 -119 [1,31oxazolidinel-2',17(2H)-dione (17a): (20 mg, 18%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.37 (dd, J, = 8.2 Hz, J2 = 17.7 Hz, 1H), 3.16 (s, 2H), 4.39 (s, 2H), 7.31 (m, 3H), 7.38 (t, J= 7.6 Hz, 1H); "C NMR (CDC 3 ): 11.4, 13.8, 20.2, 21.7, 27.8, 30.6, 31.5, 32.8, 33.8, 35.0, 35.4, 35.8, 39.4, 40.8, 47.7, 48.1, 51.3, 53.8, 55.6, 78.9, 127.9, 128.0, 128.8 (2x), 129.7, 135.9, 157.6, 221.3; LRMS for C 2 8

H

3 7

NO

3 Na [M+Na] t 458.3; HRMS calcd for C 2 8

H

38

NO

3 [M+H]f 436.2846, found 436.2854; HPLC purity: 98.7% (RT = 13.9 min; 70:30 to 5:95 MeOH/H 2 0 isocratic gradient). [003541 (3R, 5S, 8R, 9S, 10S, 13S, 14S)-10, 13-dimethyl-3'-(4 methylbenzVl)tetrahydrodecahydro-2'H-spirolcyclopenta[a lphenanthrene-3,5' [1,31oxazolidinel-2',17(2H)-dione (17b): (134 mg, 38%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 21H), 2.32 (s, 3H), 2.37 (dd, J; = 8.7 Hz, J2 = 18.2 Hz, 1H), 3.13 (s, 2H), 4.34 (s, 2H), 7.19 (s, 4H); LRMS for C 2 9

H

3 9NO 3 Na [M+Na]* 472.2. 100355] (3R, 5S, 8R, 9S, 10S, 13S, 14S)-3'-(4-methoxybenzyl)-10,13 dimethyltetradecahydro-2'H-spirocyclopenta a]lphenanthrene-3,5' [1,31oxazolidinel-2',17(2H)-dione (17c): (18 mg, 16%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.37 (dd, J = 8.7 Hz, J 2 = 18.2 Hz, IH), 3.12 (s, 2H), 3.79 (s, 3H), 4.31 (s, 2H), 6.93 (d, J= 8.7 Hz, 2H), 7.23 (d, J= 8.7 Hz, 2H); LRMS for C2 9

H

4

ONO

4 Na [M+Na]f 488.3. [00356] (3R, 5S, 8R, 9S, 10S, 13S, 14S)-10,13-dimethyl-3'-[4 (trifluoromethyl)benzvlltetradecahydro-2'H-spirolcyclopenta[alphenanthrene 3,5'-[1,3]oxazolidinel-2',17(2H)-dione (17d): (55 mg, 33%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.87 (s, 3H), 0.80-2.10 (m, 21H), 2.38 (dd, J1 = 8.8 Hz, J2 = 18.2 Hz, 1H), 3.23 (s, 2H), 4.51 (s, 2H), 7.56 (d, J= 8.0 Hz, 2H), 7.74 (d, J= 8.1 Hz, 2H); LRMS for C2 9

H

36

F

3

NO

3 Na [M+Na]+ 526.1.

WO 2012/129673 PCT/CA2012/000316 - 120 [003571 O3R. 5S, 8R. 9S. lOS, 13S, 14S)-10. 13-dimethvl-3'-I[6 trifluoromethyl)pyridine-3-ylmethyl} tetrahydrodecahydro-2 'H spirolcyclopentaial Dhenanthrene-3,5'-1 1,3loxazolidinel-2 ',17(2H)-dione (1 7e): (18 mg, 24%); 1'H NMR (Acetone-d 6 ): 0.84 (s, 3 H), 0. 87 (s, 3 H), 0. 80-2. 10 (in, 2 1H), 2.3 8 (dd, J1 = 8.8 Hz, J 2 = 18.2 Hz, I H), 3.30 (s, 2H), 4.5 8 (s, 2H), 7.87 (d, J = 8.0 Hz, I H), 8.03 (d, J = 8. 0 Hz, I1H), 8.73 (s, I1H); LRMS for C 2 8H 3 5F 3

N

2

O

3 Na [M±Na]+ 527.4. 1003581 O3R. 5, 8R. 9S, lOS, 13S, 14S)-10,13-dimethyl-3'-[2 (trifluoromethyl)benzylltetradecahydro-2'H-spirocyclopentallphenanthrene 3,5'-I1,3]oxazolidinel-2',17(2H)-dione (17f): (62 mng, 37%): 1 H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.88 (s, 3H), 0.82-2.10 (mn, 211H), 2.38 (dd, J1 = 8.7 Hz, J2 18.3 Hz, IH), 3.23 (s, 2H), 4.61 (s, 2H), 7.57 (in, 211), 7.74 (in, 2H); LRMS for C2 9

H

36

F

3

NO

3 Na [M+Na]+ 526.4. [003591 O3R, 5S, 8R. 9S. lOS. 13S. 14S)-10,13-dimethyl-3'-13 (trifluoromethyl)benzvlltetradecahydro-2'H-sirocyclopental al phenanthrene 3,5'-I1,3]oxazolidinel-2',17(2H)-dione (17g): (30 mng, 18%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.87 (s, 3H), 0.82-2.10 (in, 21H), 2.38 (dd, J 1 =8.7 Hz, J2 18.2 Hz, IH), 3.23 (s, 2H), 4.52 (s, 2H), 7.65 (mn, 4H); LRMS for C 29

H

36

F

3

NO

3 Na [M+Naf 526.0. [003601 O3R, 5S, 8R, 9S, lOS. 13S, 14S)-3'-13,5 bis(trifluoromethvl)benzvI1l-10,13-dimethytetradecahydro-2'H spirolcyclonenta i £11henanthrene-3,5'- [1 ,3]oxazolidinel-2 ',17(2H)-dione (17h): (28 mg, 15%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H-), 0.87 (s, 3H), 0.81-2.10 (mn, 21H), 2.38 (dd, J 1 = 8.7 Hz, J 2 = 17.7 Hz, 1H), 3.31 (s, 2H), 4.64 (s, 2H-), 8.00 (s, 3H-); LRMS calcd for C 3 oH 35

F

6

NO

3 Na [M+Na]+ 594.2.

WO 2012/129673 PCT/CA2012/000316 - 121 [00361] (3R, 5S, 8R, 9S, 1OS, 13S, 14S)-3'-cyclohexyl-10,13 dimethyltetradecahvdro-2'H-spiro[cyclopenta Ial phenanthrene-3,5' [1,31oxazolidinel-2',17(2H)-dione (17): (19 mg, 18%); 1H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.88 (s, 3H), 0.80-2.08 (m, 31H), 2.38 (dd, J/ = 8.8 Hz, J 2 = 18.3 Hz, 1H), 3.23 (s, 2H), 3.53 (m, 1H); LRMS for C 27

H

4 1

NO

3 Na [M+Na]f 450.4. 100362] Synthesis of (3R, 5S, 8R, 9S, 10S, 13S, 14S)-10,13,16,16 tetramethyl-3'-(4-methylbenzvl)tetrahydrodecahvdro-2'H spirofcvclopentafalphenanthrene-3,5 '-1,3oxazolidineI-2',1 7(2H)-dione (18) [00363] To a solution of compound 17b (100 mg, 0.22 mmol) in anhydrous THF (15 mL), under an argon atmosphere, was added NaH (60% in oil; 89 mg, 2.2 mmol). The resulting solution was stirred at room temperature over a period of I h. Methyl iodide (110 gL, 250 mg, 1.76 mmol) was then added and the solution refluxed overnight. The reaction mixture was subsequently poured into water and extracted three times with EtOAc. The combined organic layers were washed with brine and dried with MgSO 4 . The crude compound was purified by flash chromatography using EtOAc/hexanes (2:8) as the eluant system to provide compound 18 (32 mg, 30%). 'H NMR (CDCl 3 ): 0.77 (s, 3H), 0.87 (s, 3H), 1.03 (s, 3H), 1.16 (s, 3H), 0.80-1.85 (in, 20H), 2.34 (s, 3H), 3.03 (s, 2H), 4.37 (s, 2H), 7.15 (s, 4H); LRMS for C 31

H

43

NO

3 Na [M+Na]+ 500.2. [003641 Synthesis of (3a, 5c)-3-{[(2{[tert-butyl(dimethyl)silylloxy} ethyl)aminolmethyll-3-hydroxyandrostan-17-one (19) [00365] To a solution of oxirane 650 in anhydrous ethanol (50 mL) was added 2 -{[tert-butyl(dimethyl)silyl] oxylethanolamine (1.75 g, 10.0 mmol) and the solution refluxed over a period of 5 h. The resulting solution was subsequently concentrated and purified by flash chromatography using EtOAc/hexanes (2:8) as the eluent system to provide compound 19 (1.0 g, 66%). 'H NMR (Acetone-d 6 ): 0.08 (s, WO 2012/129673 PCT/CA2012/000316 - 122 6H), 0.81 (s, 6H), 0.91 (s, 9H), 0.80-2.10 (in, 20H), 2.22 and 2.38 (2m, 2H), 2.48 (s, 2H), 2.72 (t, J= 5.5 Hz, 2H), 3.25 (in, I H), 3.72 (t, J= 5.5 Hz, 2H), 3.80 (t, J= 6.0 Hz, 1H); LRMS calcd for C 2 8H 52

NO

3 Si [M+H]* 478.2. [00366] Synthesis of (3R, 5S, 8R, 9S, 10S, 13S, 14S)-3'(2-{[tert butyl(dimethyl)silylloxylethyl)-10,13-dimethyltetrahydrodecahydro-2'H spiroicyclopenta alphenanthrene-3,5'-[1,31oxazolidinel-2',17(2H)-dione (20) 1003671 To a solution of compound 19 (1.0 g, 2.2 mmol) in anhydrous DCM (100 mL), at 0 0 C and under an argon atmosphere, was added diisopropylamine (760 ptL, 564 mg, 4.4 mmol) and triphosgene (325 mg, 1.1 mmol). The solution was subsequently stirred over a period of 8 h while at room temperature. The reaction mixture was then poured into water, extracted twice with DCM, filtered using a phase separator (Biotage, Uppsala, Sweden) and concentrated. The crude compound was purified by flash chromatography using EtOAc/hexanes (2:8) as the eluant system to provide compound 20 (370 mg, 36%). 'H NMR (Acetone-d 6 ): 0.09 (s, 6H), 0.85 (s, 3H), 0.89 (s, 3H), 0.91 (s, 9H), 0.80-2.07 (in, 22H), 2.38 (dd, J = 8.6 Hz, J 2 = 18.3 Hz, 1H), 3.30 (t, J = 5.3 Hz, 2H), 3.40 (s, IH), 3.78 (t, J= 5.4 Hz, 2H); LRMS for C2 9

H

49

NO

4 SiNa [M+Na]* 526.5. [00368] Synthesis of (3R, 5S, 8R, 9S, 1OS, 13S, 14S)-3'-(2-hydroxyethybI 10,13-dimethyltetrahydrodecahydro-2'H-spirofcyclopenta~alphenanthrene-3,5' {1,3|oxazolidinel-2 ',17(2H)-dione (21) [003691 To a solution of compound 20 (360 mg, 0.76 mmol) in anhydrous THF (70 mL), at room temperature and under an argon atmosphere, was added a solution of tetrabutylammonium fluoride (TBAF) in THF (1.0 M; 1.05 mL, 1.5 mmol). The solution was stirred at room temperature over a period of I h. The resulting reaction mixture was then poured into water and extracted twice with EtOAc. The combined organic layers were dried with Na 2

SO

4 , filtered and evaporated WO 2012/129673 PCT/CA2012/000316 - 123 under reduced pressure to provide crude 21. The crude compound was purified by flash chromatography using EtOAc to provide pure 21 (225 mg, 76%). 'H NMR (Acetone-d 6 ): 0.85 (s, 3H), 0.89 (s, 3H), 0.80-2.08 (m, 23H), 2.37 (dd, J = 8.6 Hz, J 2 = 18.3 Hz, I H), 3.29 (t, J= 5.3 Hz, 2H), 3.40 (s, 1 H), 3.67 (t, J= 5.6 Hz, 2H); LRMS for C 23

H

35

NO

4 Na [M+Na]f 412.2. 100370] Synthesis of (3R, 5S, 8R, 9S, 1OS, 13S, 14S)-3'-(2-bromoethyl) 10,13-dimethyltetrahydrodecahydro-2'H-spiro[cyclopentalalphenanthrene-3,5' [1,31oxazolidinel-2',17(2H)-dione (22) [00371] To a solution of compound 21 (225 mg, 0.58 mmol) in anhydrous DCM (50 mL), at 0 0 C and under an argon atmosphere, was added triphenylphosphine (303 mg, 1.16 mmol) and carbon tetrabromide (383 mg, 1.16 mmol). The solution was stirred at room temperature over a period of I h. The resulting reaction mixture was then washed with water and the organic phase dried using a phase separator syringe (Biotage). The crude compound was purified by flash chromatography using EtOAc/hexanes (4:6) as the eluant system to provide compound 22 (235 mg, 90%). 1H NMR (Acetone-d 6 ): 0.85 (s, 3H), 0.89 (s, 3H), 0.82-2.08 (m, H, residual CH and

CH

2 ), 2.38 (dd, J 1 = 8.6 Hz, J 2 = 18.3 Hz, 1H), 3.41 (s, 2H), 3.63 (s, 4H); LRMS for

C

23

H

34 79 BrNO 3 Na [M+Na]* 474.1. [003721 Synthesis of (3R, 5S, 8R, 9S, 1OS, 13S, 14S)-10,13-dimethyl-3'

(

2

-(

3 -methylphenoxv)ethylltetrahydrodecahydro-2'H spirofcyclopentafalphenanthrene-3,5 '-[1,3toxazolidinel-2',1 7(2H)-dione (23a) [00373] To a solution 3-methylphenol (22 pL, 22 mg, 0.200 mmol) and

K

2

CO

3 (28 mg, 0.200 mmol) in anhydrous DMF (2 mL), stirred over a period of 10 min at 70'C, was subsequently added compound 22 (30 mg, 0.067 mmol). The reaction mixture was stirred overnight under an argon atmosphere at 70 0 C. The reaction mixture was then poured into an aqueous NaOH solution (1.0 N) and WO 2012/129673 PCT/CA2012/000316 - 124 extracted three times with diethylether. The combined organic layers were successively washed with brine, dried over Na 2

SO

4 , filtered, and concentrated. The crude product was purified by flash chromatography using EtOAc/hexanes (2:8) as the eluant system to provide compound 23a (11 mg, 34%). 1 H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.88 (s, 3H), 0.80-2.07 (m, H, residual CH and CH 2 ), 2.29 (s, 3H), 2.37 (dd, J = 8.7 Hz, J 2 = 18.8 Hz, 1H), 3.45 (s, 2H), 3.59 (m, 2H), 4.15 (t, J= 5.3 Hz, 2H), 6.77 (in, 3H), 7.16 (t, J = 7.8 Hz, IH); LRMS for C 3 0

H

4 1

NO

4 Na [M+Na]f 502.4. [00374] Synthesis of (3R, 5S, SR, 9S, loS, 13S, 14S)-10,13-dimethyl-3' {2-(3-methylphenyl)sulfonyll ethyl)tetrahydrodecahydro-2'H spiro[cyclopentalalphenanthrene-3,5'-[1,31oxazolidinel-2',17(2H)-dione (23b) 100375] To a solution 3-methylbenzenthiol (41 mg, 0.33 mmol) and

K

2

CO

3 (46 mg, 0.33 mmol) in anhydrous DMF (3 mL), stirred over a period of 10 min at 70'C, was subsequently added compound 22 (50 mg, 0.11 mmol). The reaction mixture was stirred over a period of 3 h under an argon atmosphere at 70'C. The reaction mixture was then poured into water and extracted three times with diethylether. The combined organic layers were successively washed with brine, dried over Na 2

SO

4 , filtered, and concentrated to provide the corresponding crude thioether derivative. The crude thioether product (45 mg) was subsequently diluted in a methanol/water mixture (1:1) followed by the addition of oxone (112 mg). The resulting reaction mixture was then stirred overnight while at room temperature. The crude reaction mixture was subsequently poured into water and extracted three times with EtOAc. The combined organic layers were then washed with brine, dried over Na 2

SO

4 , filtered, and concentrated. The crude product was purified by flash chromatography using EtOAc/hexanes (1:1) as the eluant system to provide compound 23b (9 mg, 16% for the combined two-step reaction). 'H NMR (Acetone d 6 ): 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.07 (m, 21H), 2.38 (dd, J = 8.7 Hz, J 2 = 8.3 Hz, I H), 2.46 (s, 3H), 3.30 (s, 2H), 3.50 (t, J= 6.6 Hz, 2H), 3.61 (m, 2H), 7.57 (m, 2H), 7.77 (m, 2H); LRMS for C 3 oH 4 jNOSSNa [M+Na]* 550.2.

WO 2012/129673 PCT/CA2012/000316 - 125 [003761 General Procedure for the Synthesis of 3-Carbamate androsterone derivatives 23c-23f [003771 To a solution of compound 22 (30 mg, 0.067 mmol) in anhydrous DMF (2 mL) was added the appropriate secondary amine (0.20 mmol) and sodium carbonate (21 mg, 0.20 mmol). The reaction mixture was stirred over a period of 3 h at 70'C. The reaction mixture was then poured into water and extracted three times with diethylether. The combined organic layers were successively washed with brine, dried over Na 2

SO

4 , filtered, and concentrated. The crude product was purified by flash chromatography using either dichloromethane/methanol/triethylamine (90:9:1) as the eluant system to provide pure 23c, 23e and 23f or EtOAc/hexanes (1:1) as the eluant system to provide 23d. 100378] (3R, 5S, 8R, 9S, 10S, 13S, 14S)-10,13-dimethyl-3'-12 (piperidin-1-yl)ethylltetrahydrodecahydro-2'H spirolcyclopenta lal phenanthrene-3,5'-[1,31oxazolidinel-2', 1 7(2H)-dione (23c): (7 mg, 22%); 1H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.89 (s, 3H), 0.80-2.08 (m, 24H), 2.26 (in, 2H), 2.38 (dd, J = 8.8 Hz, J 2 = 18.3 Hz, 1H), 3.05-3.30 (in, 4H), 3.42 (s, 2H), 3.46 (t, J = 6.5 Hz, 2H), 3.80 (t, J = 6.5 Hz, 2H); LRMS for C 28

H

47

N

3 0 3

[M+NH

3 ]* 473.3. [00379] (3R, 5S, 8R, 9S, 10S, 13S, 14S)-3'-{2 lethyl(phenyl)aminoIethyl}-10,13-dimethyltetrahydrodecahydro-2'H spiro[cyclopentalalphenanthrene-3,5'-[1,31oxazolidinel-2',17(2H)-dione (23d): (16 mg, 42%); 'H NMR (Acetone-d 6 ): 0.85 (s, 3H), 0.88 (s, 3H), 0.82-2.07 (in, 24H), 1.03 (t,J= 7.1 Hz, 3H), 2.38 (dd,J = 8.7 Hz,J 2 = 18.3 Hz, 1H), 2.53 (q,J= 7.1 Hz, IH), 2.60 (t, J= 6.1 Hz, 2H), 3.28 (s, 2H), 3.32 (in, 2H), 3.59 (s, 2H), 7.23 (t, J= 7.2 Hz, I H), 7.30 (t, J= 7.3 Hz, 2H), 7.36 (t, J= 7.3 Hz, 2H); LRMS for C 32

H

4 6

N

2

O

3 Na [M+Na]f 529.4.

WO 2012/129673 PCT/CA2012/000316 - 126 100380] (3R, 5S, 8R, 9S, 10S, 13S, 14S)-3'-{2 [cyclohexyl(ethyl)aminolethyl}-10,13-dimethyltetradecahydro-2'H spiro IcyclopentaJal phenanthrene-3,5'-[1,31oxazolidinel-2',17(2H)-dione (23e): (13 mg, 39%); 'H NMR (CDC1 3 ): 0.81 (s, 3H), 0.86 (s, 3H), 1.41 (t, J= 7.2 Hz, 3H), 2.00 (s, 2H), 0.80-2.40 (in, 30H), 2.43 (dd, J1 = 8.6 Hz, J 2 = 19.3 Hz, IH), 3.45 (in, 4H), 3.63 (in, IH), 3.72 (in, 1H), 3.85 (in, 2H); LRMS calcd for C 31

H

53

N

3 0 3

[M+NH

3 ]* 515.2. [00381] (3R, 5S, 8R, 9S, 1OS, 13S, 14S)-3'-[2-(dipropylamino)ethyll 10,13-dimethyltetradecahydro-2'H-spirocyclopentalalphenanthrene-3,5' 11,3]oxazolidinel-2',17(2H)-dione (23f): (13 mg, 40%); 'H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.89 (s, 3H), 0.78-2.10 (in, 28H), 0.94 (t, J= 7.4 Hz, 3H), 2.38 (dd, J; = 8.7 Hz, J 2 = 18.2 Hz, 1 H), 3.23 (in, 4H), 3.42 (s, 2H), 3.48 (t, J= 6.6 Hz, 2H), 3.74 (t, J = 6.9 Hz, 2H); LRMS calcd for C 29 H5 1

N

3 0 3

[M+NH

3 ]+ 489.3. [003821 Synthesis of N-{(3R, 5S, 8R, 9S, 1OS, 13S, 14S)-10,13-dimethyl 2 ',17-dioxohexadecahydro-3'H-spiro~cyclopenta~alphenanthrene-3,5' (1,31oxazolidine-3'-vllethyll-N-propylpropanamide (232) [003831 To a solution of compound 22 (30 mg, 0.067 mmol) in anhydrous DMF (3 mL) was added K 2

CO

3 (21 mg, 0.20 mmol) and N-propylamine (16 pL, 12 mg, 0.20 mmol). The reaction mixture was stirred over a period of 5 h at 80'C. The reaction mixture was then poured into water and extracted three times with diethylether. The combined organic layers were successively washed with brine, dried over Na 2

SO

4 , filtered, and concentrated. The crude secondary amine product was used in the next step without further purification. [00384] To a solution of the crude secondary amine product (30 mg) in anhydrous dichloromethane (50 mL), were successively added triethylamine (27 ptL, 21 mg) and propionyl chloride (13 pL, 13 mg). The solution was stirred at room WO 2012/129673 PCT/CA2012/000316 - 127 temperature over a period of 4 h while under an argon atmosphere. The reaction mixture was then diluted with dichloromethane (15 mL), washed with water, dried using a phase separator syringe (Biotage), and concentrated. The resulting crude product was purified by flash chromatography using EtOAc/hexanes (1:1) as the eluant system to provide pure 23g (11 mg, 34%). 1 H NMR (Acetone-d 6 ): 0.84 (s, 3H), 0.88 (s, 3H), 0.91 (t, J = 7.4 Hz, 3H), 1.04 (t, J= 7.4 Hz, 3H), 0.82-2.07 (in, 23H), 2.34 (in, 3H), 3.25-3.40 (in, 6H), 3.53 (in, 2H); LRMS for C 29 H4 6

N

2 0 4 Na [M+Na]* 509.1. 1003851 Biological Assays 1003861 Inhibition of 1 7fl-HSD3 in homogenated cells [00387] The inhibitory activity of the compounds was determined following a known literature.

48

'

53 Briefly, HEK-293 cells overexpressing 17P-HSD3 were homogenated and used for the enzymatic assay. The transformation of 50 nM of [4- 14C]-4-androstene-3,17-dione ([14C]-A4 -dione) into [14Cl-testosterone ( 14C]-T) in presence of excess NADPH (5 mM) and ethanol (control) or an inhibitor dissolved in ethanol was measured and used to determine the percentage of inhibition at a given concentration. 1003881 Inhibition of 170-HSD-3 in intact cells 1003891 HEK-293 cells were seeded at 200000 cells/well in a 12-well plate (BD Falcon) at 37'C under a 95% air, 5% CO 2 humidified atmosphere in minimum essential medium (MEM) containing non-essential amino acids (0.1 mM), glutamine (2 mM), sodium pyruvate (1 mM), 10% foetal bovine serum, penicillin (100 IU/mL) and streptomycin (100 ptg/mL). The expression vector encoding for 17p-HSD3 was transfected following the Exgen 500 procedure (Fermentas, Burlington, ON, Canada) with 2 pg of recombinant plasmid per well. For the inhibitory activity assay, a final WO 2012/129673 PCT/CA2012/000316 - 128 concentration of 50 nM of [4-' 4 C]-4-androstene-3,17-dione in ethanol (53.6 mCi/mmol, Perkin Elmer Life Sciences Inc., Boston, MA, USA) and an ethanolic solution of inhibitor (0.5% v/v) were added to freshly culture medium and incubated over a period of I h. Each inhibitor was assessed in triplicate at tested concentrations. After incubation, the culture medium was removed and the radiolabeled steroids extracted and quantified as described for the enzymatic assay in homogenated cells.

48 Percentage transformation and the IC 50 values were calculated as previously reported.

48 100390] Proliferative activity on androgen-sensitive (AR*) Shionogi cells 100391] The proliferative (androgenic) activity was determined using androgen-sensitive Shionogi cells following a known literature procedure.

5 4 Inhibitors and reference compounds were tested at two concentrations (0.1 and 1.0 iM). The androgenicity was reported as % cell proliferation (versus the control). The basal cell proliferation (control) was fixed at 100%. [003921 General procedure for the synthesis of 3A-3D (Scheme 19) 100393] In a Schlenk tube, oxirane 2 (1.7 mmol) was dissolved in dry MeOH (15 mL), followed by the addition of the appropriate amino acid methyl ester (17.2 mmol). The Schlenk tube was then hermetically closed and heated at 90'C with stirring over a period of 21 h. The MeOH was then evaporated and the crude reaction mixture dissolved in DCM. The solution was then pre-adsorbed on silica gel and purified by flash column chromatography (Hexanes / EtOAc / TEA (89:10:1). 1003941 3 p-Methyl-N-[(17,17-diethylendioxy-3a-hydroxy-5a androstane)l-methyl-L-Leucinate (3A) WO 2012/129673 PCT/CA2012/000316 -129 [003951 Yield: 99%; Rf = 0.6 (hexanes/EtOAc, 1:1); IR (film): 3479 and 3333 (OH and NH), 1736 (C=O); 'H NMR (Acetone-d 6 ): 0.74 (m, I H), 0.78 (s, CH 3 19), 0.82 (s, 3H, CH 3 -18), 0.91 and 0.92 (2d, J = 6.9 Hz, (CH 3

)

2 - from iPr), 1.10-1.95 (unassigned CH and CH 2 ), 2.21 and 2.57 (2d of AB system, J = 11.6 Hz, CH 2 N), 3.14 (s, OH), 3.23 (dd, J = 6.4 Hz, J = 8.2 Hz, CHC=O) 3.67 (s, OCH), 3.83 (m, OCH 2 CH 2 O); 1 3 C NMR (CDCl 3 ): 11.20, 14.38, 20.33, 21.92, 22.64, 22.87, 24.82, 28.51, 30.72, 31.23, 31.67, 33.77, 34.18, 35.79, 35.96, 38.58, 40.58, 42.75, 45.96, 50.29, 51.67, 53.87, 59.32, 60.87, 64.52, 65.12, 69.90, 119.48, 176.32. [00396] 3p-Methyl-N-1(17,17-diethylendioxy-3a-hydroxy-5ci androstane)]-methyl-D-Leucinate (3B) [003971 Yield: 99%; Rf = 0.3 (hexanes/EtOAc, 1:1); IR (film): 3479 and 3333 (OH and NH), 1736 (C=0); 'H NMR (CDCl 3 ): 0.73 (s, CH 3 -19), 0.83 (s, CH 3 18), 0.90 and 0.93 (2d, J = 6.6 Hz, (CH 3

)

2 - from iPr), 0.96-1.97 (unassigned CH and

CH

2 ), 2.17 and 2.62 (2d of AB system, J = 11.9 Hz, CH 2 N), 3.04 (s, OH), 3.21-3.25 (tapp, J = 6.7 Hz, CHC=O), 3.72 (s, OCH 3 ), 3.87 (m, OCH 2

-CH

2 0); 13 C NMR (CDC1 3 ): 11.16, 14.35, 20.33, 21.91, 22.61, 22.84, 24.78, 28.44, 30.68, 31.16, 31.54, 33.63, 34.13, 35.76, 35.93, 38.56, 40.56, 42.70, 45.90, 50.25, 51.61, 53.82, 59.31, 60.83, 64.47, 65.07, 69.91, 119.41, 176.28. [00398] 3p-Methyl-N-1(17,17-diethylendioxy-3a-hydroxy-5a androstane)]-methyl-L-phenylalaninate (3C) 100399] Yield: 77%; Rf = 0.5 (hexanes/EtOAc, 1:1); IR (film): 3472-3340 (NH and OH), 3024 (CH, Ph), 1736 (C=O); 'H NMR (Acetone-d 6 ): 0.75 (s, CH 3 -19), 0.82 (s, CH 3 -18), 0.95 (m, IH), 1.01-1.97 (unassigned CH and CH 2 ), 2.24-2.56 (2d of AB system, J = 11.6 Hz, CH 2 N), 2.92 (m, CH 2 -Ph), 3.04 (s, OH), 3.43-3.45 (tapp, J = 6.6 Hz, CHC=O), 3.62 (s, OCH 3 ), 3.84 (m, OCH 2

-CH

2 O); 7.25 (m, Ph); 1 3 C NMR (CDCl 3 ): 11.19, 14.39, 20.33, 22.65, 28.49, 30.71, 31.22, 31.53, 33.67, 34.18, 35.78, WO 2012/129673 PCT/CA2012/000316 - 130 35.93, 38.50, 39.81, 40.49, 45.95, 50.27, 51.72, 53.81, 59.34, 63.92, 64.51, 65.11, 69.97, 70.03, 119.45, 126.74, 128.42, 129.10, 137.28, 175.02. [004001 3p-Methyl-N-1(17,17-diethylendioxy-3a-hydroxy-5a androstane)]-methyl-D-phenylalaninate (3D) 1004011 Yield: 87%; Rf = 0.43 (hexanes/EtOAc, 1:1); IR (film): 3472 3340 (NH and OH), 3024 (CH, Ph), 1736 (C=O); 'H NMR (Acetone-d 6 ): 0.75 (s,

CH

3 -19), 0.82 (s, CH 3 -18), 0.95 (in, 1H), 1.50-1.97 (unassigned CH and CH 2 ), 2.24 2.56 (2d of AB system, J = 11.6 Hz, CH 2 N), 2.92 (in, CH 2 -Ph), 3.04 (s, OH), 3.43 3.45 (tapp, J = 6.6 Hz, CHC=O), 3.62 (s, OCH 3 ), 3.84 (in, OCH 2

-CH

2 0); 7.25 (in, Ph); "C NMR (CDC1 3 ): 11.17, 14.39, 20.35, 22.65, 28.43, 30.71, 31.18, 31.49, 33.61, 34.17, 35.77, 35.93, 38.41, 39.79, 40.47, 45.93, 50.26, 51.70, 53.80, 59.33, 63.96, 64.50, 65.10, 69.97, 70.02, 119.43, 126.72, 128.40, 129.10, 137.31, 175.03. [004021 Synthesis of 3p-Methyl-N-[(17,17-diethylendioxy-3a-hydroxy Sa-androstane)]-methyl glycinate (3E) [00403] Anhydrous methanol (20 mL) was added to a mixture of glycine methyl ester hydrochloride (1.1 g, 8.8 mmol) and DIPEA (2.2 g, 17.5 mmol) placed in a Schlenk tube. The resulting solution was subsequently stirred over a period of 30 minutes while at room temperature. Oxirane 2 (0.3 g, 0.9 mmol) was then added and the resulting solution heated at 95'C over a period of 22 h. The solution was then cooled to room temperature and filtered. The resulting filtrate was then concentrated and the residue (1.8 g) dissolved in DCM. The solution was then pre-adsorbed on silica gel and purified by flash column chromatography (hexanes / EtOAc (7:3); 1% TEA). The title compound was isolated as a yellowish product (3E). Yield: 53%; Rf = 0.17 (hexanes/EtOAc, 1:1); IR (film): 3464-3348 (NH and OH), 1736 (C=O); 1H NMR (CDCl 3 ): 0.74 (s, CH 3 -19), 0.83 (s, CH 3 -18), 0.85-1.97 (unassigned CH and

CH

2 ), 2.50 (s, CH 2 N), 3.45 (s, NCH 2 C=O), 3.73 (s, OCH 3 ), 3.83-3.95 (in, OCH 2 CH 2 O); 1 3 C NMR (Acetone-d 6 ): 10.75, 13.91, 20.26, 22.44, 30.65, 31.38, 31.46, WO 2012/129673 PCT/CA2012/000316 - 131 33.34, 33.71, 33.91, 35.79, 35.90, 38.48, 40.45, 45.77, 50.32, 50.73, 51.18, 54.43, 61.09, 64.23, 64.83, 69.95, 118.87, 172.74. [00404] General Procedure for the Synthesis of Azaspirolactones 4A-4E (Scheme 19) [004051 To a solution of MeONa (0.37 mmol) in anhydrous THF (24 mL) at 0 0 C was added, under argon atmosphere, a solution of the appropriate amino alcohol (3A-3E) (0.61 mmol) in anhydrous THF (28 mL). The resulting solution was stirred over a period of 2 h at room temperature. The reaction was subsequently quenched with a saturated ammonium chloride solution. The crude product was extracted with EtOAc (4 x 50 mL), the combined organic layers dried with anhydrous MgSO 4 and concentrated. The residue was dissolved in DCM and the solution pre adsorbed on silica gel and purified by flash column chromatography (hexanes / EtOAc (8:2); 1% TEA). 1004061 Azaspirolactone 4A: Yield: 89%; Rf = 0.49 (hexanes/ethyl acetate 1:1); 'H NMR (Acetone-d 6 ): 0.79 (m, IH), 0.83 (s, CH 3 -19 and CH 3 -18), 0.89 and 0.92 (2d, J = 6.6 Hz, (CH 3

)

2 - from iPr), 0.95-1.98 (unassigned CH and CH 2 ), 2.83 and 2.93 (2d of AB system, J = 13.5 Hz, CH 2 N), 3.43 (m, NCHC=O), 3.85 (m, OCH 2 CH 2 O). [00407] Azaspirolactone 4B: Yield: 84%; Rf = 0.49 (hexanes/ethyl acetate 1:1); 'H NMR (CDCl 3 ): 0.76 (s, 3H, CH 3 -19), 0.83 (s, 3H, CH 3 -18), 0.91-0.97 (2d, J = 6.3 Hz, 3H, (CH 3

)

2 - from iPr), 2.78-2.89 (dd, J = 13.5 Hz, 2H, CH 2 N), 3.41 3.50 (m, 1H, NCHC=O), 3.83-3.94 (m, 4H, OCH 2

-CH

2 0); "C NMR (CDCI 3 ): 11.33, 14.38, 20.34, 20.94, 22.66, 23.40, 24.46, 28.24, 30.64, 31.49, 32.63, 34.18, 35.74, 36.03, 38.31, 39.25, 39.65, 41.46, 45.93, 50.13, 52.55, 53.38, 55.54, 64.54, 65.14, 82.29, 119.39, 171.91.

WO 2012/129673 PCT/CA2012/000316 - 132 [004081 Azaspirolactone 4C: Yield: 62%; Rf = 0.3 (hexanes/ethyl acetate 1:1); 1 H NMR (Acetone-d 6 ): 0.78 (s, 3H, CH 3 -19), 0.82 (s, 3H, CH 3 -18), 2.86-2.99 (m, 2H, CH 2 N), 3.01-3.20 (m, 2H, CH 2 Ph), 3.70-3.74 (m, IH, NCHC=O), 3.79-3.89 (m, 4H, OCH 2

-CH

2 0), 7.22-7.30 (2m, 5H, Ph). [00409] Azaspirolactone 4D: Yield: 65%; Rf = 0.25 (hexanes/ethyl acetate 1:1); 'H NMR (Acetone-d 6 ): 0.77 (s, 3H, CH 3 -19), 0.82 (s, 3H, CH 3 -18), 2.76 2.90 (m, 2H, CH 2 N), 3.02-3.16 (m, 2H, CH 2 Ph), 3.72-3.75 (m, IH, NCHC=O), 3.78 3.89 (m, 4H, OCH 2

-CH

2 0), 7.22-7.32 (2m, 5H, Ph). 100410] Azaspirolactone 4E: Yield: 52%; Rf = 0.27 (hexanes/ethyl acetate 1:1); 'H NMR (CDC 3 ): 0.78 (s, 3H, CH 3 -19), 0.83 (s, 3H, CH 3 -18), 2.82 (s, 2H, CH 2 N), 3.63 (s, 2H, NCH 2 C=O), 3.85-3.95 (m, 4H, OCH 2

-CH

2 0); 1 3 C NMR (CDC1 3 ): 11.34, 14.39, 20.36, 22.65, 28.14, 30.63, 30.97, 31.25, 32.69, 34.18, 35.74, 36.00, 37.96, 39.39, 45.93, 47.55, 50.13, 53.05, 53.38, 64.55, 65.15, 82.52, 119.39, 168.78. [004111 General Procedure for the Synthesis of N-benzylated Azaspirolactones 6A-6E (Scheme 20) [00412] In a Schlenk tube, the appropriate azaspirolactone (4A-4E) (0.1 mmol) was dissolved in dry DCM (5 mL), followed by the drop-wise addition of diisopropylethylamine (0.17 mmol). The Schlenk tube was then hermetically closed and heated at 75'C with stirring over a period of 10 min. The Schlenk tube was then cooled to room temperature followed by the addition of benzyl bromide (1.7 mmol). The reaction mixture was subsequently stirred and heated at 75'C for an additional 48 h. After cooling the Schlenk tube, silica gel was added and the mixture concentrated. The residue was purified by flash column chromatography (hexanes / EtOAc (8:2); 1% TEA).

WO 2012/129673 PCT/CA2012/000316 - 133 [004131 N-Benzylated Azaspirolactone 6A: Yield: 63%; Rf = 0.83 (hexanes/ethyl acetate 1:1); 'H NMR (Acetone-d 6 ): 0.74 (s, 3H, CH 3 -19), 0.81 (s, 3H,

CH

3 -18), 0.92-0.99 (2d, J = 6.7 Hz, 6H, (CH 3

)

2 - from iPr), 2.22-2.76 (dd, J = 12.5 Hz, 2H, CH 2 N), 3.19-4.10 (dd, J =13.7 Hz, 2H, CH 2 -Ph), 3.15-3.18 (m, IH, NCHC=O), 3.78-3.88 (m, 4H, OCH 2

-CH

2 0), 7.28-7.40 (m, 5H, Ph). 100414] N-Benzylated Azaspirolactone 6B: Yield: 49%; Rf = 0.83 (hexanes/ethyl acetate 1:1); 'H NMR (CDCl 3 ): 0.68 (s, 3H, CH 3 -19), 0.82 (s, 3H,

CH

3 -18), 0.94-0.99 (2d, J = 6.7 Hz, 3H, (CH 3

)

2 - from iPr), 2.16-2.65 (dd, J= 12.4 Hz, 2H, CH 2 N), 3.11-4.04 (dd, J =13.5 Hz, 2H, CH 2 -Ph), 3.15-3.20 (m, IH, NCHC=O), 3.83-3.94 (m, 4H, OCH 2

-CH

2 O), 7.29-7.38 (m, 5H, Ph). 1 3 C NMR (CDC1 3 ): 11.37, 14.37, 20.31, 22.09, 22.10, 22.67, 23.93, 25.02, 26.17, 28.34, 30.65, 31.06, 31.95, 32.69, 34.19, 35.72, 36.02, 38.33, 38.59, 39.24, 39.78, 45.95, 50.14, 53.39, 57.84, 58.15, 63.04, 64.56, 65.14, 81.33, 119.41, 127.37, 128.50, 128.53, 137.78, 171.37. [004151 N-Benzvlated Azaspirolactone 6C: Yield: 70%; Rf = 0.75 (hexanes/ethyl acetate 1:1); 1H NMR (Acetone-d 6 ): 0.62 (s, 3H, CH 3 -19), 0.79 (s, 3H,

CH

3 -18), 2.20-2.59 (dd, J = 12.5 Hz, 2H, CH 2 N), 2.81-4.41 (dd, J =13.6 Hz, 2H,

NCH

2 -Ph), 3.25-3.47 (m, 2H, CH 2 Ph); 3.50-3.52 (m, IH, NCHC=O), 3.80-3.89 (m, 4H, OCH 2

-CH

2 O), 7.29-7.38 (m, IOH, Ph). 1 3 C NMR (Acetone-d 6 ): 10.73, 13.88, 20.16, 22.38, 27.86, 28.36, 29.52, 30.56, 30.64, 31.16, 33.10, 33.88, 35.24, 35.65, 35.72, 37.91, 39.53, 45.74, 50.17, 54.04, 57.45, 57.72, 60.95, 64.24, 64.83, 65.89, 80.54, 118.82, 126.42, 127.11, 127.79, 128.34, 128.53, 130.39, 137.98, 138.09, 169.53. [00416] N-Benzylated Azaspirolactone 6D: Yield: 61%; Rf = 0.75 (hexanes/ethyl acetate 1:1); 'H NMR (Acetone-d 6 ): 0.62 (s, 3H, CH 3 -19), 0.78 (s, 3H,

CH

3 -18), 2.19-2.60 (dd, J = 12.4 Hz, 2H, CH 2 N), 2.81-4.43 (dd, J =13.8 Hz, 2H,

NCH

2 -Ph), 3.27-3.49 (m, 2H, CH 2 Ph); 3.49-3.51 (m, IH, NCHC=O), 3.77-3.88 (m, 4H, OCH 2

-CH

2 O), 7.25-7.34 (m, IOH, Ph). 1 3 C NMR (Acetone-d 6 ): 10.76, 13.87, 20.18, 22.42, 27.58, 28.36, 29.52, 30.57, 31.14, 31.19, 32.69, 33.91, 35.04, 35.65, WO 2012/129673 PCT/CA2012/000316 - 134 37.26, 39.94, 45.73, 50.19, 54.10, 57.48, 57.83, 64.24, 64.81, 65.83, 80.46, 118.82, 126.54, 127.12, 127.77, 128.35, 128.59, 130.50, 137.88, 138.07, 169.43. [00417] N-Benzylated Azaspirolactone 6E: Yield: 70%; Rf = 0.83 (hexanes/ethyl acetate 1:1); 'H NMR (CDC1 3 ): 0.72 (s, 3H, CH 3 -19), 0.82 (s, 3H,

CH

3 -18), 2.41 (s-broad, 2H, CH 2 N); 3.26 (s, 2H, NCH 2 C=O), 3.83-3.94 (in, 4H,

OCH

2

-CH

2 0), 7.26-7.36 (in, 5H, Ph). 1 3 C NMR (CDC1 3 ): 11.4, 14.39, 20.33, 22.66, 28.12, 30.65, 31.01, 31.84, 32.90, 34.19, 35.74, 36.03, 38.43, 39.57, 45.94, 50.14, 53.40, 55.42, 59.79, 60.99, 61.36, 64.55, 65.15, 82.49, 119.40, 127.60, 128.53, 128.71, 136.69, 168.28. 100418] General Procedure for the Synthesis of Azaspirolactones 5A-5E and N-benzylated Azaspirolactones 7A- 7E (Scheme 20) [004191 To a solution of the appropriate azaspirolactone (4A-4E) or N benzylated azaspirolactone (6A-6E) (30 mg to 60 mg) in dioxane (2 mL) was added an aqueous sulfuric acid solution (5%; 2 mL) and the resulting reaction mixture stirred at room temperature over a period ranging from 2-5 h. The progress of the reaction was monitored by TLC. An aqueous saturated solution of sodium bicarbonate (12 mL) was then added to the reaction mixture which was subsequently extracted four times with ethyl acetate (4 x 12 mL). The combined organic layers were dried with anhydrous MgSO 4 and concentrated. The crude compound was purified by flash chromatography using either hexanes/EtOAc (9:1) + 1% TEA (compounds 5A-5E) or hexanes/EtOAc (95:5) + 1% TEA (compounds 7A-7E) as the eluant system. [00420] Azaspirolactone 5A: Yield: 71%; IR (film) v 3448-3333 (NH), 1736 (C=O), 1458 (C-0) cm-1; 'H NMR (Acetone-d 6 ): 0.84 (s, 3H, CH 3 -19), 0.85 (s, 3H, CH 3 -18), 0.89-0.93 (2d, J = 6.6 Hz, 6H, (CH 3

)

2 - from iPr), 2.80-2.96 (dd, J = 13.5 Hz, 2H, CH 2 N), 3.41-3.44(2d, J = 4.1 Hz, lH, NCHC=O); 1 3 C NMR (Acetone-d 6 ) 10.73, 13.16, 20.13, 20.62, 21.44, 22.86, 24.15, 27.88, 30.66, 31.16, 31.70, 33.16, WO 2012/129673 PCT/CA2012/000316 - 135 34.97, 35.10, 36.05, 37.91, 39.81, 41.29, 47.28, 51.25, 51.94, 54.28, 55.45, 60.95, 80.81, 170.77. [00421] Azaspirolactone 5B: Yield: 92%; IR (film): 3448-3340 (NH), 1736 (C=O), 1458 (C-0); 'H NMR (CDC1 3 ): 0.80 (s, 3H, CH 3 -19), 0.86 (s, 3H, CH 3 18), 0.92-0.997 (2d, J = 6.4 Hz, 6H, (CH 3

)

2 - from iPr), 2.80-2.90 (dd, J = 13.5 Hz, 2H,

CH

2 N), 3.41-3.44(2d, J = 3.70 Hz and J = 3.43, IH, NCHC=O); "C NMR (CDC1 3 ): 11.35, 13.82, 20.22, 20.95, 21.75, 23.40, 24.47, 28.02, 30.53, 31.44, 31.51, 32.60, 35.00, 35.83, 36.12, 38.24, 39.66, 41.39, 47.76, 51.37, 52.48, 53.78, 55.58, 82.23, 171.81, 221.23. [004221 Azaspirolactone 5C: Yield: 97%; IR (film): 3333 (NH), 3032 (CH, Ph), 1736 (C=0), 1450 (C-0); 'H NMR (Acetone-d 6 ): 0.81 (s, 3H, CH 3 -19), 0.84 (s, 3H, CH 3 -18), 2.77-2.91 (dd, J = 13.3 Hz, 2H, CH 2 N), 2.99-3.19 (m, 2H,

CH

2 Ph), 3.72-3.75 (2d, J = 4.0 Hz, IH, NCHC=O), 7.22-7.30 (m, 5H, Ph); "C NMR (CDCl 3 ): 11.30, 13.82, 20.22, 21.74, 27.82, 30.51, 30.99, 31.50, 32.83, 35.01, 35.83, 36.02, 37.98, 39.20, 47.76, 51.38, 52.62, 53.73, 53.76, 58.59, 58.73, 82.72, 127.08, 128.77, 129.52, 137.17, 170.77, 221.27. [00423] Azaspirolactone 5D: Yield: 56%; IR (film): 3448-3325 (NH), 3024 (CH, Ph), 1728 (C=O), 1450 (C-0); 'H NMR (Acetone-d 6 ): 0.80 (s, 3H, CH 3 19), 0.83 (s, 3H, CH 3 -18), 2.77-2.89 (dd, J = 13.3 Hz, 2H, CH 2 N), 3.03-3.16 (m, 2H,

CH

2 Ph), 3.73-3.76 (2d, J = 4.1 Hz, IH, NCHC=O), 7.23-7.30 (m, 5H, Ph); "C NMR (CDCl 3 ): 11.31, 13.81, 20.21, 21.75, 27.78, 30.54, 31.27, 31.51, 32.51, 34.99, 35.84, 36.03, 37.95, 39.59, 47.75, 51.38, 52.66, 53.76, 58.75, 60.82, 82.66, 127.09, 128.77, 129.59, 130.48, 137.23, 170.70, 221.22. 100424] Azaspirolactone 5E: Yield: 54%; Rf = 0.29 (hexanes/ethyl acetate 1:1); IR (film): 3448-3333 (NH), 1736 (C=O), 1450 (C-0); 'H NMR (CDCl 3 ): 0.80 (s, 3H, CH 3 -19), 0.86 (s, 3H, CH 3 -18), 2.83 (s, 2H, CH 2 N), 3.63 (s, 2H,

NCH

2 C=O); "C NMR (CDC1 3 ): 11.36, 13.83, 20.23, 21.75, 27.90, 30.53, 31.19, WO 2012/129673 PCT/CA2012/000316 - 136 31.49, 32.66, 35.00, 35.83, 36.08, 37.88, 39.40, 47.55, 47.6, 51.35, 52.93, 53.78, 82.45, 168.69, 221.27. 1004251 N-Benzylated Azaspirolactone 7A: Yield: 81%; Rf = 0.77 (hexanes/ethyl acetate 1:1); IR (film): 3448 (NH), 1736 (C=0), 1450 (C-0); 'H NMR (Acetone-d 6 ): 0.77 (s, 3H, CH 3 -19), 0.83 (s, 3H, CH 3 -18), 0.92-0.99 (2d, J = 6.3 Hz and J = 6.5 Hz, 6H, (CH 3

)

2 - from iPr), 2.26-2.77 (dd, J = 12.5 Hz, 2H, CH 2 N), 3.15 3.18 (2d, J= 2.6 and J = 2.7, 1 H, NCHC=O), 3.19-4.10 (dd, J =13.6 Hz, 2H, CH 2 -Ph), 7.28-7.40 (m, 5H, Ph); 1 3 C NMR (Acetone-d 6 ): 10.81, 13.19, 20.13, 21.44, 21.64, 23.44, 24.85, 27.77, 30.64, 31.52, 31.69, 33.25, 34.94, 35.11, 36.03, 38.15, 38.33, 39.74, 47.29, 51.25, 54.26, 57.63, 57.72, 63.07, 80.48, 127.18, 128.38, 128.57, 128.62, 138.20, 205.18, 205.24, 218.67. 100426] N-Benzylated Azaspirolactone 7B: Yield: 92%; Rf = 0.85 (hexanes/ethyl acetate 1:1); IR (film): 3448 (NH), 3032 (CH, Ph), 1736 (C=0), 1450 (C-O); 'H NMR (CDC1 3 ): 0.71 (s, 3H, CH 3 -19), 0.84 (s, 3 H, CH 3 -18), 0.92-0.99 (2d, J = 6.5 Hz and J = 6.7 Hz, 6H, (CH 3

)

2 - from iPr), 2.17-2.66 (dd, J = 12.4 Hz, 2H,

CH

2 N), 3.11-4.04 (dd, J =13.5 Hz, 2H, CH 2 -Ph), 3.15-3.18 (2d, J = 2.8 Hz and J = 3.0 Hz, 1H, NCHC=O), 7.29-7.36 (m, 5H, Ph); "C NMR (CDC] 3 ): 11.39, 13.82, 20.19, 21.76, 22.08, 23.95, 25.01, 28.11, 30.63, 31.51, 31.89, 32.65, 34.98, 35.84, 36.10, 38.22, 38.47, 39.80, 47.76, 50.14, 51.40, 53.81, 57.73, 58.13, 63.05, 64.56, 65.14, 127.40, 128.52, 128.55, 137.74, 171.30, 221.25. [004271 N-Benzvlated Azaspirolactone 7C: Yield: 70%; Rf = 0.23 (hexanes/ethyl acetate 1:1); IR (film): 3441 (NII), 3032 (CH, Ph), 1728 (C=O), 1450 (C-O); 1 H NMR (Acetone-d 6 ): 0.65 (s, 3H, CH 3 -19), 0.80 (s, 3H, CH 3 -18), 2.20-2.60 (dd, J = 12.5 Hz, 2H, CH 2 N), 3.26-4.42 (dd, J =13.9 Hz, 2H, NCH 2 -Ph), 3.31-3.43 (m, 2H, CH 2 Ph); 3.47-3.53 (m, 1H, NCHC=O), 7.26-7.36 (m, IOH, Ph); "C NMR (Acetone-d 6 ): 10.68, 13.14, 20.03, 21.41, 27.71, 30.60, 30.62, 31.64, 33.02, 34.91, 35.09, 35.19, 35.82, 37.85, 39.55, 47.27, 51.21, 54.13, 57.42, 57.72, 65.88, 80.50, WO 2012/129673 PCT/CA2012/000316 - 137 126.45, 127.11, 127.79, 128.35, 128.53, 130.39, 137.96, 138.06, 169.50, 205.17, 205.25, 218.69. 1004281 N-Benzylated Azaspirolactone 7D: Yield: 81%; IR (film): 3448 (NH), 3032 (CH, Ph), 1728 (C=O), 1450 (C-0); 1 H NMR (Acetone-d 6 ): 0.65 (s, 3H,

CH

3 -19), 0.80 (s, 3H, CH 3 -18), 2.20-2.61 (dd, J = 12.4 Hz, 2H, CH 2 N), 3.27-4.44 (dd, J =13.8 Hz, 2H, NCH 2 -Ph), 3.38-3.43 (m, 2H, CH 2 Ph); 3.49-3.51 (m, IH, NCHC=O), 7.26-7.34 (m, IOH, Ph); 1 3 C NMR (Acetone-d 6 ): 10.74, 13.15, 20.06, 21.44, 27.42, 30.57, 31.15, 31.67, 32.62, 34.90, 35.04, 35.12, 35.75, 37.18, 39.96, 47.27, 51.23, 54.22, 57.41, 57.84, 60.95, 65.84, 80.45, 126.55, 127.14, 127.77, 128.37, 128.60, 130.54, 137.88, 138.08, 169.42, 205.28, 218.72. [00429] N-Benzylated Azaspirolactone 7E: Yield: 71%; Rf = 0.7 (DCM/MeOH 39:1); IR (film): 3448 (NH), 1720 (C=0), 1450 (C-0); 'H NMR

(CDC

3 ): 0.75 (s, 3H, CH 3 -19), 0.85 (s, 3H, CH 3 -18), 2.42 (s-broad, 2H, CH 2 N); 3.27 (s, 2H, NCH 2 C=O), 3.52-3.53 (d, J= 4.4 Hz, 2H, CH 2 -Ph), 7.29-7.37 (m, 5H, Ph); 1 3 C NMR (CDC1 3 ): 11.41, 13.82, 20.21, 21.75, 27.90, 30.57, 31.51, 31.77, 32.86, 35.01, 35.84, 36.12, 38.34, 39.59, 47.76, 51.38, 53.81, 55,42, 59.69, 61.36, 82.38, 127.62, 128.54, 128.72, 136.65, 168.19, 221.26. [00430] General procedure for the synthesis of carbamates 9A-9E (Scheme 21) 1004311 To a solution of the appropriate amino alcohol 3 (0.12 mmol) in DCM (3 mL) at 0 0 C and under an argon atmosphere, was added diisopropylethylamine (0.24 mmol). The reaction mixture was then stirred over a period of 10 min followed by the addition of triphosgene (0.06 mmol). The resulting reaction mixture was then stirred over a period of 5 h while at room temperature. An acidic solution composed of HCI / MeOH (10:90) (2 mL) was then added and the resulting reaction mixture stirred overnight. The reaction was subsequently quenched by the addition of a saturated aqueous solution of NaHCO 3 (10 mL). The desired WO 2012/129673 PCT/CA2012/000316 - 138 product was subsequently extracted with dichloromethane (4 x 10 mL). The combined organic layers were dried using anhydrous Na 2

SO

4 and concentrated. The crude compound was purified by flash chromatography using hexanes / EtOAc (95:5) + 1% TEA as the eluant system. [004321 Carbamate 9A: Yield: 73%; Rf = 0.71 (hexanes/ ethyl acetate 1:1); IR (film): 3464 (NH), 1744 (C=O), 1435 (C-0); 'H NMR (CDCl 3 ): 0.82 (s, 3H,

CH

3 -19), 0.85(s, 3H, CH 3 -18), 0.96-0.98 (2d, J = 1.8 Hz and J = 2.0 Hz, 6H, (CH 3

)

2 from iPr), 3.12-3.44 (dd, J = 8.1 Hz, 2H, CH 2 N), 3.72 (s, 3H, OCH 3 ), 4.56-4.60 (2d, J = 4.8 Hz, I H, NCHCO); 3 C NMR (CDCl 3 ): 11.39, 13.82, 20.23, 21.08, 21.75, 23.15, 24.94, 27.88, 30.59, 31.47, 32.73, 33.81, 35.01, 35.41, 35.83, 37.71, 39.47, 40.77, 40.84, 47.74, 51.31, 52.22, 52.88, 53.58, 53.88, 79.64, 157.58, 171.99, 221.17. 100433] Carbamate 9B: Yield: 67%; Rf = 0.57 (hexanes/ ethyl acetate 1:1); IR (film): 3464 (NH), 1736 (C=0), 1443 (C-0); 'H NMR (CDCl 3 ): 0.83 (s, 3H,

CH

3 -19), 0.86 (s, 3H, CH 3 -18), 0.96-0.98 (d, J = 6.5 Hz, 6H, (CH 3

)

2 - from iPr), 3.13 3.45 (dd, J = 8.1 Hz, 2H, CH 2 N), 3.72 (s, 3H, OCH 3 ), 4.58-4.62 (2d, J = 4.8 Hz, IH, NCHCO); 1 3 C NMR (CDCl 3 ): 11.40, 13.80, 20.22, 21.07, 21.72, 23.11, 24.93, 27.81, 30.56, 31.47, 32.88, 33.86, 35.00, 35.37, 35.81, 37.69, 39.28, 40.77, 40.83, 47.71, 51.30, 52.18, 52.83, 53.56, 53.86, 79.64, 157.53, 171.97, 221.13. 1004341 Carbamate 9C: Yield: 61%; Rf = 0.40 (hexanes/ ethyl acetate 1:1); IR (film): 3464 (NH), 3032 (CH, Ph) 1744 (C=0), 1435 (C-0); 'H NMR (CDC1 3 ): 0.76 (s, 3H, CH 3 -19), 0.84 (s, 3H, CH 3 -18), 2.92-3.38 (2m, 2H, CH 2 Ph), 3.08-3.32 (dd, J = 8.0 Hz, 2H, CH 2 N), 3.76 (s, 3H, OCH 3 ), 4.86-4.90 (2 d, J= 5.5 Hz, IH, NCHCO); "C NMR (CDCl 3 ): 11.33, 13.80, 20.19, 21.72, 27.71, 27.79, 30.50, 31.45, 32.44, 33.80, 34.96, 35.07, 35.29, 35.81, 38.80, 40.56, 47.71, 51.28, 52.42, 53.20, 53.83, 55.81, 79.71, 127.07, 128.52, 128.58, 128.71, 136.02, 157.31, 171.00, 221.16.

WO 2012/129673 PCT/CA2012/000316 - 139 1004351 Carbamate 9D: Yield: 75%; Rf = 0.50 (hexanes/ ethyl acetate 1:1); IR (film): 3456 (NH), 1736 (C=O), 1435 (C-0); 'H NMR (CDCl 3 ): 0.77 (s, 3H,

CH

3 -19), 0.84 (s, 3H, CH 3 -18), 2.92-3.41 (2m, 2H, CH 2 Ph), 3.09-3.32 (dd, J = 8.0 Hz, 2H, CH 2 N), 3.76 (s, 3H, OCH 3 ), 4.84-4.89 (2 d, J = 5.5 Hz, 1 H, NCHCO); 1 3 C NMR (CDCl 3 ): 11.35, 13.80, 20.19, 21.72, 27.71, 27.79, 30.54, 31.46, 32.35, 33.72, 34.98, 35.07, 35.30, 35.82, 39.14, 40.78, 47.71, 51.29, 52.41, 53.32, 53.83, 55.91, 79.67, 127.09, 128.52, 128.59, 128.71, 136.02, 157.29, 171.02, 221.16. [00436] Carbamate 9E: Yield: 32%; Rf = 0.48 (hexanes/ ethyl acetate 1:1); IR (film): 3464 (NH), 1744 (C=O), 1443 (C-0); 'H NMR (CDCl 3 ): 0.82 (s, 3H,

CH

3 -19), 0.85 (s, 3H, CH 3 -18), 3.33 (s, 2H, CH 2 N), 3.75 (s, 3H, OCH 3 ), 4.01-4.02 (d, J = 4.33 Hz, IH, NCHCO); 13 C NMR (CDC 3 ): 11.42, 13.82, 20.23, 21.74, 27.83, 30.58, 31.48, 32.69, 33.84, 35.01, 35.37, 35.83, 39.27, 40.82, 45.01, 47.73, 51.31, 52.26, 53.89, 56.52, 79.61, 157.57, 169.02, 221.17. [004371 17D-estradiol (170-E2); 17a-estradiol (17c-E2); 18-epi-17p E2; and 18-epi-17a-E2; 1004381 Two E2 isomers with the 18-methyl group inversed (18-epi), compounds 3 and 4 (Scheme 22A), were prepared and their estrogenic activity tested in representative in vitro and in vivo assays.

55 WO 2012/129673 PCT/CA2012/000316 - 140 12 18 OH OH 11 17 1 10 9 H 13 16 H 2 8H 141 5 H H HO 3 6 HO 17a-E2 (2) 17p-E2 (1) OH OH H H H H H H HO HO 18-epi-17p-E2 (3) 18-epi-17a-E2 (4) Scheme 22A [00439] [2,4,6,7 3 H]-17p-estradiol was obtained from American Radiolabeled Chemicals (St.Louis, MO, USA). Natural estradiol (17P-E2; 1) and the corresponding 17a-isomer (17a-E2; 2) were purchased from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada). 18-Epi- 17p3-E2 (3) and 18-epi- I 7a-E2 (4) were prepared from (13a)-3-hydroxyestra-1(10),2,4-trien-17-one (18-epi-estrone). The purity of compounds 1-4 was determined by high performance liquid chromatography (HPLC) using a Waters' apparatus (Waters Associates Milford, MA, USA) having a Waters 996 Photodiode array detector (207 nm), a Phenyl/hexyl-RP column (75 x 4.6 mm id, 3 pm) from Phenomenex (Torrance, CA, USA) and a linear solvent gradient from 60:40 to 95:5 of methanol/water. [004401 Synthesis of 18-epi-1 71-E2 and 18-epi-1 7a-E2 1004411 To a solution of 18-epi-estrone (125 mg, 0.46 mmol) in anhydrous THIF (5 mL) was added LiAIH 4 (2.3 mL of a 1.0 M solution in THF) at room temperature. The solution was stirred over a period of 90 min while under an argon atmosphere. The resulting solution was then poured into a saturated Rochelle salt WO 2012/129673 PCT/CA2012/000316 - 141 solution (50 mL) and then extracted twice with EtOAc. The combined organic layers were washed with brine, dried with Na 2

SO

4 , filtered and concentrated to provide 120 mg of a mixture of alcohols. Purification with a Biotage flash chromatography system (Uppsala, Sweden) using a solvent gradient from EtOAc/Hexanes 1:9 to 3:7 and a silica gel column (KP-Sil, 60A) provided two fractions. The first fraction was shown to contain 18-epi-17p-E2 (3) (35 mg, 28% yield) with an HPLC purity of 99.7%. The other fraction (33 mg, 27% yield) was shown to contain 18-epi-17a-E2 (4) with an HPLC purity of 96.5% in addition to 2.8% of undesired 17p-E2 (1). To ensure the absence of 17p-E2, this fraction was recrystallized from acetonitrile (1% w/v) to provide 17 mg of compound 4 with an HPLC purity of 99.7%. [00442] (13ac,17 )-estra-1(10),2,4-triene-3,17-diol (3): HPLC purity: 99.7%; RT = 9.2 min (Gradient from 60:40 to 95:5 of methanol/water, Phenyl/hexyl RP column (75 x 4.6 mm id, 3 pum; Phenomenex); LRMS for C 1 gH 26 0 2 Na [M + Na]*: 295.4 m/z or for C 18

H

25 0 2 (M-H)-: 271.4 m/z; 1H NMR (CD 3 OD): 0.96 (s, 18-CH 3 ), 1.10-2.15 (residual CH and CH 2 ), 2.28 (in, 6-CH 2 ), 2.72 (m, 2H), 3.76 (dd, J, = 4.0 Hz and J 2 = 6.0 Hz, 17ax-H), 6.46 (d, J = 2.3 Hz, 4-CH), 6.55 (dd, J, = 2.4 Hz and J 2 = 8.4 Hz, 2-CH), 7.07 (d, J = 8.5 Hz, 1-CH); 1 3 C NMR (CD 3 0D): 26.1 (C15), 28.7 (ClI), 28.9 (C7), 29.0 (C18), 30.1 (C6), 30.9 (C12), 31.9 (C16), 40.0 (C9), 42.0 (C8), 44.0 (C13), 51.5 (C14), 82.6 (C17), 112.6 (C2), 114.2 (C4), 127.0 (Cl), 132.2 (C1O), 137.7 (C5), 154.2 (C3); NOESY showed correlations between 14a-CH and 18-CH 3 as well as 18-CH 3 and 17-CH, thus demonstrating the 18-epi-CH 3 and 17a-CH configurations of 3 (18-epi-l 7p-E2). [004431 (13at,17a)-estra-1(10),2,4-triene-3,17-diol (4): HPLC purity: 99.7%; RT = 7.9 min (Gradient from 60:40 to 95:5 of methanol/water, Phenyl/hexyl RP column (75 x 4.6 mm id, 3 pm; Phenomenex); LRMS for CisH 26 0 2 Na [M + Na]*: 295.4 m/z or for C 18

H

2 5 0 2 [M-H] : 271.3 m/z; IH NMR (CD 3 0D): 0.92 (s, 18-CH 3 ), 0.95-2.30 (residual CH and CH 2 ), 2.72 (in, 6-CH 2 ), 4.19 (t, J = 8.5 Hz, 17p-H), 6.50 (d, J = 2.6 Hz, 4-CH), 6.57 (dd, J 1 = 2.6 Hz, J 2 = 8.4 Hz, 2-CH), 7.12 (d, J = 8.5 Hz, 1-CH); 1 3 C NMR (CD 3 0D): 22.0 (C18), 23.5 (C15), 26.5 (Cl1), 28.5 (C7), 28.7 WO 2012/129673 PCT/CA2012/000316 - 142 (C16), 30.1 (C6), 32.8 (C12) , 42.3 (C9), 42.7 (C8), 43.2 (C13), 50.3 (C14), 73.1 (C17), 112.5 (C2), 114.5 (C4), 126.4 (Cl), 131.1 (CIO), 137.8 (C5), 154.5 (C3); NOESY showed a correlation between l4a-CH and 18-CH 3 , but no correlation between 18-CH 3 and 17-CH, thus demonstrating the 18-epi-CH 3 and 170-CH configurations of 4 (1 8-epi- 1 7a-E2). 1004441 In Vitro Estrogenic Activity [004451 Cell Culture Maintenance [00446] Human breast cancer cell lines (T-47D, MCF-7 and BT-20) were obtained from the American Type Culture Collection (ATCC) and maintained in culture flasks (75 cm 3 growth area) at 37 0 C under a 5% CO 2 humidified atmosphere. The T-47D cells were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 ptg/mL) and 17P-E2 (1 nM). The MCF-7 cells were propagated in Dubelcco's Modified Eagle's Medium nutrient mixture F-12 Ham (DMEM-F12) medium supplemented with 5% FBS, glutamine (2 nM), penicillin (100 IU/mL), streptomycin (100 ptg/mL) and 173-E2 (1 nM). BT-20 cells were grown in minimal essential medium (MEM) supplemented with 10% (v/v) FBS, glutamine (2 nM), penicillin (100 UI/mL) and streptomycin (100 pg/m L).

WO 2012/129673 PCT/CA2012/000316 - 143 [004471 Cell Culture Assay [004481 The cells from each breast cancer cell line were seeded into 96 well plates (3000 cells per well). The cells were suspended in the appropriate culture medium reported above, except that FBS was replaced by 5% (v/v) FBS treated with dextran-coated charcoal to remove the endogenous steroids and the medium was supplemented with insulin (50 ng/mL). After 48 h of deprivation, the cells were incubated for 7 days at 37*C in presence of 17p-E2 (1), 17a-E2 (2), 18-epi-17p-E2 (3) and 18-epi-17a-E2 (4) at different concentrations in freshly changed medium. The effects of the drugs on the growth of three different cell lines (MCF-7, T47-D and BT 20) were determined by using 20 ptL of 3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent (Owen's reagent, Cell Titer 96@, Aqueous One Solution, Promega, USA). MTS was added to each well and the reaction was stopped after 4 h. The reagent is converted to water soluble colored formazan by dehydrogenase enzymes present in metabolically active cells. The ability of cells to cleave MTS is indicative of the degree of mitochondrial/cellular respiration within those cells. Subsequently, the absorbance was recorded at 490 nm with a 96 well plate reader (Molecular Devices, Sunnyvale, CA, USA). The conversion of MTS by untreated cells at the end of the cultured period was set at 100%. The results shown are representative of two separate experiments run in triplicate. [004491 Estrogen ReceDtor (ER) Binding [004501 Tissue Preparation of ER [00451] Female Sprague-Dawley rats, weighing 200-300 g were obtained from Charles-River (St. Constant, QC, Canada). The rats were gonadectomised under general anaesthesia (Isoflurane) and killed by cervical dislocation 24 h later. The uteri were rapidly removed, dissected free from adhering tissue and frozen on dry-ice and kept at -80'C before their use. All subsequent steps needed for the ER preparation WO 2012/129673 PCT/CA2012/000316 - 144 were performed at -4 0 C. Uteri were homogenized in 10 volume (w/v) of buffer A (25 mM Tris-HCI, 1.5 mM EDTA disodium salt, 10 mM at-monothioglycerol, 10% glycerol, and 10 mM sodium molybdate, pH 7.4), using a Polytron PT-10 homogenizer (Brinkman Instruments, Canada) at a setting of 5 for three periods of 10 s, with intervals of 10 s for cooling. The homogenate was then centrifuged at 105 000 x g over a period of 60 min in a Beckman L5-65 ultracentrifuge (Fullerton, USA). 1004521 ER Bindin2 Assay 100453] Estrogen binding was measured using the dextran-coated charcoal adsorption technique. Radioactive 17p-E2 ([ 3 H]-17p-E2) solubilised in ethanol was diluted into buffer A. Aliquots of uterine cytosol preparation (0.1 mL) were incubated with 5 nM of [3 H]-17p-E2 (approximately 200 000 cpm, 0.1 mL) in the presence or absence of the indicated concentrations of compounds 1-4 (0.1 mL, prepared in buffer A containing 10% of ethanol) for 3 h at room temperature. Unbound steroids were then separated by incubation for 15 min at room temperature with 0.3 mL 0.5% Norit A and 0.005% Dextran T-70 in buffer B (1.5 mM EDTA disodium salt, 10 mM a monothioglycerol, and 10 mM Tris-HCL, pH 7.4) and centrifuged at 3000 x g for 15 min. Aliquots of the supernatant (0.3 mL) were removed for radioactivity measurement. After the addition of 10 mL of Formula-989 scintillation liquid (New England Nuclear-DuPont), the radioactivity was measured in a Beckman counter at a counting efficiency of 62%. The relative binding affinity (RBA) of the tested compounds was calculated as IC 50 ([3H]-17p-E2/iC50 (tested compound) X 100.

WO 2012/129673 PCT/CA2012/000316 - 145 [00454] In Vivo Estrogenic Activity (uterotrophic assay) [004551 Female BALB/c mice (42-53 days) weighing 18 g were obtained from Charles River (St. Constant, QC, Canada) and housed four to five per cage in temperature (22 3C) and light (12 h/day, light on at 7hl 5) controlled environment. The mice were fed rodent chow and tap water ad libitum. The animals were ovariectomized (OVX) under isoflurane-anaesthesia via bilateral flank incisions and randomly assigned to groups (5 animals by group). Mice in the OVX control group received the vehicle alone (8% ethanol-0.4% methylcellulose) during the 7-day period. The possible estrogenic activity of tested compounds was evaluated after their administration by subcutaneous (s.c.) injection [1, 10 and 100 pg/kg, s.c., twice daily (BID)] as suspension in 8% ethanol-0.4% methylcellulose to OVX female mice for 7 days. On day 8, the mice were sacrificed by cervical dislocation. The uteri and vagina were rapidly removed, freed from fat and connective tissue and weighed. Results are the means ± SEM of 5 mice per group. 1004561 Statistical Analysis [004571 Data are expressed as the means ± SEM, and statistical significance was determined according to the multiple range test of Duncan-Kramer. P values which were less than 0.05 were considered as statistically significant. 1004581 Cell Proliferation Assay [004591 ER+ Cell Lines 100460] The proliferative activity of the four isomeric compounds 1-4 was evaluated on the human breast cancer (ER*) cell lines MCF-7 and T-47D. These cell lines were chosen because they express the ER, predominantly ERa, and they proliferate in presence of estrogenic compounds. They are therefore good in vitro WO 2012/129673 PCT/CA2012/000316 - 146 models to evaluate the effect of the structural modifications of the E2 nucleus on the ER. The assay was performed at concentrations ranging from 0.02 nM to 50 1 M for each compound and the results were expressed as the percentage of cell proliferation (FIG. 14A and 15A). The cell proliferation without compounds was fixed as 100%. The four E2 isomers clearly modulated the proliferation of MCF-7 and T-47D cells, which are both sensitive to ER. However, they are some differences in the pattern of activity according to the range of concentrations and two kinds of proliferation effects, estrogenic and cytotoxic, were observed at low and high concentrations, respectively. At lower concentrations (0.01 - 5 pM), all tested compounds induced cell growth in different degrees until to reach a plateau (approximately at 200% of cellular proliferation). As expected, 17P-E2 (1) was the most estrogenic compound of the four E2-isomers in MCF-7 cells. Compound 2, with the OH in position 17a, was 45 fold less potent than natural 17p-E2 (1). Compound 3, with an inversion of 18 methyl in position 13 of 17p3-E2, was 1000 folds less estrogenic than 17p-E2 (1). Finally, with two changes in the structure of 17p-E2 nucleus, an inversion of both 17 OH and 18-methyl groups, compound 4 was surprisingly only 111 folds weaker estrogenic than 17p-E2 (1). The same tendency was observed in T-47D cells, compounds 2, 3 and 4 were 16, 125 and 55-folds less estrogenic than compound 1, respectively. In summary, the four E2 isomers induced cell proliferation on ER+ cells in the following order: 1 (17P-E2) > 2 (17a-E2) > 4 (18-epi-17a-E2) > 3 (18-epi-17p E2). After exposure of ER+ cells at high concentrations (over 5 pM) of compounds 1 4, an important cytotoxic effect in terms of cell growth inhibition was observed in both cell lines. Thus, compounds 2 and 4 seem to be more cytotoxic than 1 and 3 in MCF-7 cells (FIG. 14B) whereas compound 4 is more cytotoxic in T-47D cells (FIG. 15B). The results obtained from these cell proliferation experiments assessed the role of 18-methyl group orientation (P or a) on the estrogenic activity and cytotoxicity of natural potent estrogens 17p-E2 (1) and 17a-E2 (2). [00461] ER~ Cell Lines WO 2012/129673 PCT/CA2012/000316 - 147 [00462] The BT-20 cells are negative for estrogen receptor (ER-), but do express an estrogen receptor mRNA that has deletion of exon 5. It was decided to test compounds 1-4 in this cell line to demonstrate that the effect observed in ER' cell lines was due to the action on the estrogen receptor. When compounds 1-4 were tested on BT-20 (ER-) cells, no proliferative effect was observed at all tested concentrations (FIG. 16). At high concentrations (> I pM), cytotoxic effects were observed in BT-20 cells. This effect on ER- cells was observed in the same range of concentrations than for ER' cell lines (FIG. 16). The present data demonstrate that the cytotoxic activity at high concentrations is working by a non ER-dependent mechanism. 1004631 ER-Binding Affinity [004641 Having assessed the in vitro estrogenic activity for the four E2 isomers, the affinity of compounds 1-4 for ER was assessed next. A binding assay using ER obtained from uteri of gonadectomised rats was performed. Since the predominant isoform in the mature uterus is ERa, as in the MCF-7 cells, the results were expressed as a function of the affinity for the ERa. The concentration at which the unlabeled ligand displaces half the specific binding of [ 3 H]-1 7p-E2 to ER (ICso) was determined by computer fitting of the data using non-linear regression analysis. Compounds 1-4 bound to the ER in different degree as represented by the dose response curves (FIG. 17). The relative binding affinity (RBA) of natural 170-E2 (1) for the ER was established as 100%, because it had the highest ER-binding affinity for ER of the four E2 isomers. Compound 2 (17a-E2) had a RBA of 3.6% indicating that the inversion of 17p-OH to 17a-OH decreases the affinity for ER of 28 folds. The RBA of compounds 3 and 4 were 1.2% and 1.6%, respectively, thus showing a weak ER binding affinity. These two compounds share an important modification of the structure of natural estrogen 17p-E2 (1), the inversion of 18-methyl in position 13, which is apparently responsible for the major lost, 81 and 66 folds, of binding affinity for the ER. The ranking order is thus 17p-E2 (1)> 17a-E2 (2)> 18-epi- I 7a-E2 (4) > 18-epi-17p-E2 (3) (Table 5). These results are in good agreement with the findings generated from the in vitro proliferation tests with ER+ cells.

WO 2012/129673 PCT/CA2012/000316 -148 100465] Table 5: Structural characteristic of compounds 1-4 and radiolabeled ligand assay for ER 18-CH 3 17-OH A (17-OHs) ICSO b ERa-RBA Compound orientation orientation (A) (nM) (%) 17p-E2 (1) 1 P 0 19 100 17a-E2 (2) p a 2.4 542 3.5 18-epi-17p-E2 (3) a P 2.9 1534 1.2 18-epi-17a-E2 (4) a a 1.2 1257 1.5 aA (17-OHs): Distance between the 17-OH of a given compound and the optimal OH ositioning of 170-E2 (1). The concentration of tested compound inhibiting 50% of the binding of labelled 17p-E2

(IC

50 ) was obtained from dose response curves. 'RBA: Relative Binding Affinity. [00466] Uterotrophic Activity (in vivo) 1004671 Another approach to evaluate the estrogenic activity of compounds 1-4 was to use the ovariectomized (OVX) mouse model by measuring the weight of the uterus (FIG. 18A) and the vagina (FIG. 18B), two estrogen-sensitive (ER) tissues. When 17p-E2 (1) was administrated subcutaneously (s.c.) to OVX mice, an increase of the uterine weight from 24 mg (control) to 125, 155 and 160 mg, depending on the dose (1, 10 and 100 pg/kg, respectively), was observed. For 17a-E2 (2) and 18-epi-17a-E2 (4), the increase in uterine weight was only significantly different (PS 0.01) to control group (3.6 and 2.6-folds, respectively) at a dose of 100 ig/kg suggesting an estrogenic effect. This was not in the same way for the 18-epi 17p-E2 (3) which presented a weakly and not significant uterotrophic activity (1.5 folds vs. control group) at this high dose. Doses of I and 10 jig/kg reflect the same pattern of response. Measuring the weight of vagina demonstrated the same tendency as observed with the uterus. Thus, like for the uterus, the order of estrogenic potencies in vagina was 17p-E2 (1) > 17a-E2 (2) > 18-epi-17a-E2 (4), whereas no significant uterotrophic activity was obtained with 18-epi-17p-E2 (3). Such results are in agreement with the ER binding affinity data.

WO 2012/129673 PCT/CA2012/000316 - 149 [004681 Structural Analysis 1004691 An examination of the three dimensional structures of compounds 1-4 was done using Chem3D software (FIG. 19). A superposition of the phenolic A ring of the four E2 isomers suggests that the variability in D-ring orientation and shape is compatible with receptor binding affinity and some degree of activity. In fact, it is known that subtle modifications of the E2 nucleus can modulate the response pattern within a cell. From the data it appears that changes in functional groups, and in D-ring conformation, like those produced by modifying the orientation of 17-OH and 18-methyl group, can explain the variable ER-binding affinity and estrogenic activity of compounds 1-4. As expected, the ideal position of 17-OH to get an optimal interaction with key amino acids of the ER, which resulted in the best ER binding and estrogenic potency, is that of the natural ligand of ER (1 7P-E2: compound 1). Thus, changing the 17p-OH orientation of 1 to a 17a-OH orientation (compound 2) clearly reduced both the RBA and estrogenicity. The same results were also obtained for compounds 3 and 4 having an 18-methyl orientation inversed from the beta face (natural R configuration) to the alpha face (unnatural S configuration). Such modification greatly changes the shape of the 17p-E2 and 17a-E2 nuclei and consequently the 17-OH positioning. In fact, it was observed that the distance between the optimal positioning of 17p-OH (as in compound 1) and the positioning of OH in 18-epi-1 7p-E2 (3) and 18-epi-I 7a-E2 (4) correlates with the biological activity. Thus, the further removed the 17-OH of 3 and 4 is from its positioning in compound 1 (2.9 and 1.2 A for compounds 3 and 4, respectively), the less estrogenic they are and the weaker they bind to ER (Table 5) However, to explain the difference between 2 and 4, which have roughly the same distance between their 17-OH and that of 1 (2.4 and 2.9 A, respectively), the consideration of a further parameter (i.e. steric hindrance) is required. Ring D of both C18 epimers 3 and 4 has a less planar shape than ring D of 1 and 2 limiting the access to the ER. In other words, the interference between the ER and the ring D of compounds with unnatural configuration (1 8-epi methyl) is harmful to hydrogen bond formation between key amino acids and 17-OH.

WO 2012/129673 PCT/CA2012/000316 - 150 In summary, the 17-OH group positioning and steric hindrance of 18-epi-E2 nuclei can be used to explain the great reduction of estrogenic activity. 100470] Additional Isomer of E2 [00471] The synthesis of a further E2 isomer is illustrated hereinbelow in Scheme 22B. 00 HO 1I 2 3 H2z O H O H d I i n I / F|iR HO OH H d H Hi Hi / H H HO HO

H

2 N HN 5 0 4 0 Scheme 22B: Reagents and conditions: (a) 1 ,2-phenylendiamnine, acetic acid, reflux, 5 h; (b) 3-carboxamide-benzaldehyde, KOH, EtOH, rx; (c), H 2 , Pd/C (10%), MeOH, rt; (d) NaBH 4 , MeOH/DCM (5:2), rt. 1004721 3-{(E)-[(13ac,16E)-3-hydroxy-17-oxoestra-1(10),2,4-trien-16 ylidenelmethyllbenzamide (3) [004731 To a solution of compound 2 (750 mg, 2.8 mmol) in EtOH (100 mL) was added 3-formyl-benzamide (825 mg, 5.5 mmol) and an aqueous KOH solution (10%; 10 mL). The solution was then heated at reflux over a period of 30 min. The resulting solution was then diluted with water (500 mL), neutralized with WO 2012/129673 PCT/CA2012/000316 - 15] aqueous HCl 10%, and extracted with EtOAc (3 x 150 mL). The combined organic layers were washed with brine, dried with MgSO 4 and concentrated. The crude compound was purified by flash chromatography using EtOAc/Hexanes (7:3) as the eluent system providing 598 mg (54% yield) of compound 3. 'H NMR (CD 3 OD): 1.15 (s, 18-CH 3 ), 0,65-2.69 (unassigned CH and CH 2 ), 2.98 and 3.29 (in, 15-CH 2 ), 6.42 (d, J = 2.4 Hz, 4-CH), 6.54 (dd, J2 = 2.6 Hz, Ji = 8.5 Hz, 2-CH), 7.09 (d, J = 8.6 Hz, 1-CH), 7.51 (s, '-CH), 7.59 (t, J = 7.8 Hz, 5"-CH), 7.82 (d, J = 7.8 Hz, 6"-CH), 7.91 (d, J = 7.8 Hz, 4"-CH), 8.14 (s, 2"-CH). 100474] 3-{[(13a)-3-hydroxy-17-oxoestra-1(10),2,4-trien-16 ylimethyllbenzamide (4) [004751 To a solution of compound 3 (100 mg, 0.25 mmol) in MeOH (10 mL) under an argon atmosphere was added Pd on charcoal (10%) (20 mg). The reaction vessel was flushed three times with H 2 and stirred over a period of 48 h, then filtered on celite and evaporated under reduce pressure to provide 100 mg (mixture of two diastereoisomers 16x and 16p-methyl-m-benzamide-estrone). 'H NMR (DMSO d 6 ): 0.79 (s, 18-CH 3 ), 1.07 (s, 18-CH 3 ), 0.60-3.16 (unassigned CH and CH 2 ), 6.39 (in, I H), 6.48 (in, 1H), 7.02 (d, I H), 7.34 (in, 4H), 7.67 (in, 2H), 7.91 (s, I H), 9.1 (broad s, 1H, 3-OH). [00476] 3-{[(13a)-3,17-dihydroxvestra-1(10),2,4-trien-16 ylimethyllbenzamide (5) [004771 To a solution of compounds 4 (100 mg, 0.25 mmol) in MeOH (5 mL) was added NaBHI4 (50 mg, 1.31 mmol). The solution was stirred overnight. The resulting solution was concentrated under vacuo, diluted with EtOAc (50 mL), washed with water (100 mL) and brine, dried with MgSO 4 and evaporated under reduce pressure to give 100 mg of the crude 17p-alcohols (mixture of four diastereoisomers) to be separated by LCMS-prep.

WO 2012/129673 PCT/CA2012/000316 - 152 [00478] AZALACTONE INHIBITORS OF 17p-HSD1 AND 170-HSD3 1004791 The present disclosure also includes inhibitors of both 17p-HSD1 and 17p-HSD3, which are azalactone derivatives of pregnenolone having the formula Xa or Xb: O R6 0

R

6 \ N H R7\ H Ry HO; Oij Xa Xb wherein: R6 and R 7 are independently or simultaneously H, alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl. 1004801 The synthesis of such compounds of the formula Xa or Xb is shown in Scheme 23: WO 2012/129673 PCT/CA2012/000316 - 153 0 OH 0 H a HN b NH TBSO HO (Ref: 1) (A (20R,24R) 3A (20R,24S) 2B (20R,24S) 3B (20R,24R) 0 0" dd0 d0 N I -NH H H TBSOO N.H HOZ 4A (20R,24S) SA (20R,24S) 4B (20R,24R) 5B (20R,24R) d N 0 H 6A (20R,24S) 6B (20R,24R) Scheme 23 Scheme 23: Reagents and conditions: (a) Phenylalanine methyl ester (L or D); MeOH; 90 0 C; (b) CH 3 0Na; THF; rt; (c) Benzyl bromide; DIPEA; DCM; 75 'C (Schlenk tube); (d) MeOH/HCI (95/5); THF; rt. [004811 EXPERIMENTAL 100482] General Remarks: Pregnenolone was purchased from Steraloids (Wilton, NH). (L)-phenylalanine methyl ester hydrochlorides and chemical reagents of highest purity were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada), solvents were obtained from Fisher Scientific (Montreal, QC, Canada).

WO 2012/129673 PCT/CA2012/000316 - 154 Reactions were run under an inert (argon) atmosphere in oven-dried glassware. Analytical thin-layer chromatography (TLC) was performed on 0.20-mm silica gel 60 F254 plates (Fisher Scientific), and compounds were visualized by using ammonium molybdate/sulfuric acid/water (with heating). Flash column chromatography was performed with Silicycle R10030B 230-400 mesh silica gel (Qu6bec, QC, Canada). Infrared spectra (IR) were obtained from a thin film of compound usually solubilised in CH 2 Cl 2 and deposited upon a NaCl pellet. They were recorded with a Horizon MB 3000 ABB FTIR spectrometer (ABB, Canada) and only characteristic bands are reported. Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance 400 digital spectrometer (Billerica, MA, USA) and reported in ppm; The CDC1 3 IH and 1 3 C NMR signals (7.26 and 77.00 ppm respectively) and Acetone-d 6 'H and 1 3 C NMR signals (2.05 and 28.94 ppm respectively) were used as internal references. [00483] General Procedure for the Synthesis of amino alcohols 2A and 2B [004841 The oxirane 56 1 (0.5 g, 1.1 mmol) was dissolved in dry MeOH (13 ml) and L-phenylalanine methyl ester (2.0 g, 11.2 mmol) was added in a Schlenk tube. The solution was stirred and heated at 90'C during 4 days. The reaction mixture was then dissolved in DCM, filtered and evaporated under reduced pressure. The crude reaction mixture was dissolved in DCM and pre-adsorbed on silica gel to be purified by flash column chromatography using hexanes / EtOAc (85:15 to 1:1) as eluent to give compound 2B (330 mg; 47%) as major product and compound 2A as minor product (130 mg; 19%). The starting epoxide 1 (40 mg) was also recuperated. 1004851 Methyl (2R)-2-({{2R)-2-[(3S,8S,9S,10R,13S,14S)-3-{[tert butyl(dimethyl)silylloxy}-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro-1H-cyclopenta lal phenanthren- 1 7-yll-2-hydroxypropyllamino) 3-phenylpropanoate (2A) WO 2012/129673 PCT/CA2012/000316 - 155 [00486] Rf = 0.4 (hexanes/EtOAc, 7:3); IR (film): 3560 and 3333 (OH and NH), 1732 (C=O of ester). 'H NMR (CDCl 3 ): 0.06 (s, 6H, (CH 3

)

2 Si), 0.81 (s, 3H,

CH

3 -18), 0.89 (s, 9H, (CH 3

)

3 CSi), 0.99 (s, 3H, CH 3 -19), 1.15 (s, 3H, CH 3 -21), 1.25 2.16 (unassigned CH and CH 2 ), 2.23 and 2.57 (2d of AB system, J = 11.8 Hz, 2H,

CH

2 N), 2.95 (m, 2H, CH 2 -Ph), 3.44-3.46 (tapp, J = 6.0 Hz, I H, CHC=O), 3.47, (m, 1H, CH-3), 3.68 (s, 3H, OCH 3 ), 5.31 (d, J = 5.7 Hz, IH, CH-6), 7.23 (m, 5H, Ph). "C NMR (CDCl 3 ): -4.59 (Si(CH 3

)

2 ), 13.42, 18.26 (SiC(CH 3

)

3 ), 19.41, 20.92, 22.31, 23.84, 24.98, 25.93, 31.37, 31.79, 32.07, 36.56, 37.36, 39.96, 42.59, 42.79, 50.09, 51.75, 56.95, 57.22, 57.84, 63.84, 72.61, 72.98, 121.06, 126.79, 128.46,129.12, 137.18, 141.55, 175.03. 1004871 Methyl (2S)-2-({{2R)-2-1(3S,8S,9S,10R,13S,14S)-3-{[tert butyl(dimethyl)silylloxyl-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17 tetradecahydro-1H-cyclopenta [al phenanthren-17-yll-2-hydroxypropyllamino) 3-phenylpropanoate (2B ) 1004881 Rf= 0.3 (hexanes/EtOAc, 7:3); IR (film): 3607 and 3313 (OH and NH), 1736 (C=O of ester). 'H NMR (CDCl 3 ): 0.06 (s, 6H, (CH 3

)

2 Si), 0.78 (s, 3H,

CH

3 -18), 0.89 (s, 9H, (CH 3

)

3 CSi), 0.99 (s, 3H, CH 3 -19), 1.13 (s, 3H, CH 3 -21), 1.14 2.15 (unassigned CH and CH 2 ), 2.18 and 2.67 (2d of AB system, J = 12.3 Hz, 2H,

CH

2 N), 2.94 (m, 2H, CH 2 -Ph), 3.44-3.46 (tapp, J = 6.1 Hz, 1H, CHC=O), 3.47, (m, I H, CH-3), 3.68 (s, 3H, OCH 3 ), 5.32 (d, J = 5.1 Hz, 1H, CH-6), 7.23 (m, 5H, Ph). 1 3 C NMR (CDCl 3 ): -4.59 (Si(CH 3

)

2 ), 13.46, 18.25 (SiC(CH 3 )3), 19.41, 20.92, 22.52, 23.86, 24.83, 25.93, 31.38, 31.81, 32.06, 36.57, 37.37, 39.76, 39.83, 42.45, 42.79, 50.13, 51.75, 56.82, 57.27, 58.08, 64.12, 72.60, 73.23, 121.05, 126.76, 128.44, 129.15, 137.33, 141.57, 174.97. 100489] General Procedure for the Synthesis of Azalactones 3A and 3B [004901 To a solution of sodium methoxyde (969 mg, 19 mmol) in anhydrous THF (100 mL) was added a solution of the amino alcohol 2A (590 mg, WO 2012/129673 PCT/CA2012/000316 -156 0.95 mmol) in dry THF (30 mL) under argon atmosphere. The solution was stirred during 1.5 h at room temperature, and the reaction mixture quenched with water and extracted 4 times with 130 mL of ethyl acetate. The organic layer was dried with anhydrous Na 2

SO

4 , filtered and dried under reduced pressure. The residue was adsorbed on silica gel and purified by flash chromatography using hexanes/ethyl acetate (85:15) as eluent to give compound 3B as the major product (350 mg, 63%) and 3A as minor product (157 mg, 28%). 1004911 (3S,6R)-3-benzl-6-[(30)-3-{[tert butvl(dimethyl)silylloxylandrost-5-en-17-vll-6-methvlmorpholin-2-one (3A) [004921 Rf = 0.6 (hexanes/EtOAc, 3:1); IR (film): 3441-3340 (NH), 1728 (C=O of ester). 'H NMR (CDCl 3 ): 0.05 (s, 6H, (CH 3

)

2 Si), 0.86 (s, 3H, CH 3 -18), 0.89 (s, 9H, (CH 3

)

3 CSi), 1.00 (s, 3H, CH 3 -19), 1.31 (s, 3H, CH 3 -21), 1.36-2.50 (unassigned CH and CH 2 ), 2.65 and 2.96 (2d of AB system, J = 13.2 Hz, 2H, CH 2 N), 3.19 (in, 2H, CH2-Ph), 3.46 (in, I H, CH-3), 3.62 (in, I H, CHC=O), 5.31 (d, J= 4.8 Hz, I H, CH-6), 7.30 (in, 5H, Ph). 13 C NMR (CDCl 3 ): -4.60 (Si(CH 3

)

2 ), 13.62, 18.25 (SiC(CH 3

)

3 ), 19.42, 20.83, 22.83, 23.70, 23.81, 25.92, 31.28, 31.70, 32.03, 36.54, 37.35, 38.06, 39.90, 42.76, 42.82, 50.07, 51.44, 56.82, 57.67, 58.93, 72.56, 86.90, 120.88, 126.98, 128.75, 129.49, 137.36, 141.58, 170,72. [004931 (3R,6R)-3-benzyl-6-[(3)-3-{ltert butyl(dimethyl)silylloxylandrost-5-en-17-y1l-6-methylmorpholin-2-one (3B) 1004941 Rf= 0.4 (hexanes/EtOAc, 3:1); IR (film): 3367-3028 (NH), 1713 (C=O of ester). 'H NMR (CDCl 3 ): 0.07 (s, 6H, (CH 3

)

2 Si), 0.7 (s, 3H, CH 3 -18), 0.90 (s, 9H, (CH 3

)

3 CSi), 0.98 (s, 3H, CH 3 -19), 1.35 (s, 3H, CH 3 -21), 1.36-2.50 (unassigned CH and CH 2 ), 2.72 and 3.07 (2d of AB system, J= 14.0 Hz, 2H, CH 2 N), 3.20 (in, 2H,

CH

2 -Ph), 3.50 (in, IH, CH-3), 3.80 (in, 1 H, CHC=O), 5.32 (d, J= 5.1 Hz, I H, CH-6), 7.30 (in, 5H, Ph). 1 3 C NMR (CDCl 3 ): -4.58 (Si(CH 3

)

2 ), 13.66, 18.25 (SiC(CH 3

)

3

),

WO 2012/129673 PCT/CA2012/000316 -157 19.37, 20.75, 22.62, 23.25, 23.36, 25.92, 31.31, 31.70, 32.02, 36.54, 37.30, 37.38, 39.21, 42.07, 42.74, 49.82, 50.03, 55.97, 56.04, 57.48, 72.51, 87.16, 120.90, 127.25, 128.80, 129.83, 137.27, 141.55, 171.11. [00495] Synthesis of the N-benzylated Azalactones 4A and 4B 100496] To a solution of azalactones 3A (60 mg, 0.1 mmol) or 3B (150 mg, 0.25 mmol), in dry DCM (3A: 5mL; 3B: 13 mL) was added dropwise diisopropylethylamine (1.7 eq) in a Schienk tube. The solution was stirred and heated at 75'C during 10 min and then allowed to return at room temperature. Benzyl bromide (1.7 eq) was then added to the solution and the reaction mixture was stirred and heated at 75'C during 48 h. After cooling the Schlenk tube, silica gel was added to the crude mixture and the solvent was evaporated under reduce pressure. The crude compound was purified by flash chromatography using hexanes/ethyl acetate (98:2) as eluent to give corresponding compound 4A (48 mg, 70%) or compound 4B (134 mg, 79%). 1004971 (3S,6R)-3,4-dibenzyl-6-[(3)-3-f[tert butyl(dimethyl)silyll oxyl androst-5-en-17-yll-6-methylmorpholin-2-one (4A) [00498] Yield: 70%; Rf = 0.80 (hexanes/ethyl acetate 3:1); IR (film): 1728 (C=O of ester).

1 H NMR (CDCl 3 ): 0.04 (s, 6H, (CH 3

)

2 Si), 0.77 (s, 3H, CH 3 -18), 0.88 (s, 9H, (CH 3

)

3 CSi), 0.96 (s, 3H, CH 3 -19), 0.98-1.53 (unassigned CH and CH 2 ), 1.55 (s, 3H, CH 3 -21), 1.56-2.16 (unassigned CH and CH 2 ), 2.24 and 2.40 (2d of AB system, J = 12.34 Hz, 2H, CH 2 N), 3.11 and 4.35 (2d of AB system, J = 13.56 Hz, 2H, PhCH 2 N), 3.27 and 3.56 (m, 2H, CH 2 Ph), 3.41 (m, IH, CHC=O), 3.42 (m, 1H, CH 3), 5.30 (m, IH, CH-6), 7.27 (m, 10H, Ph). 13 C NMR (CDCl 3 ): -4.60 (Si(CH 3

)

2 ), 13.42, 18.24 (SiC(CH 3

)

3 ), 19.40, 20.78, 22.76, 23.51, 23.66, 25.91, 29.69, 31.20, 31.66, 32.01, 35.69, 36.49, 37.31, 39.79, 42.74, 42.79, 50.02, 56.40, 56.76, 57.50, WO 2012/129673 PCT/CA2012/000316 - 158 58.23, 66.15, 72.57, 84.96, 120.87, 126.62, 127.35, 127.96, 128.08, 128.47, 128.51, 130.36, 137.40, 137.51, 141.55, 170.94. 100499] (3R,6R)-3,4-dibenzyl-6-[(3B)-3-fitert butyl(dimethyl)silylloxylandrost-5-en-17-yll-6-methylmorpholin-2-one (4B) 100500] Yield: 79%; Rf = 0.78 (hexanes/ethyl acetate 3:1); IR (film): 1720 (C=O of ester).

1 H NMR (CDC 3 ): 0.08 (s, 6H, (CH 3

)

2 Si), 0.77 (s, 3H, CH 3 -18), 0.89 (s, 9H, (CH 3

)

3 CSi), 0.90 (s, 3H, CH 3 -19), 0.91-1.50 (unassigned CH and CH 2 ), 1.54 (s, 3H, CH 3 -21), 1.56-2.16 (unassigned CH and CH 2 ), 2.23 and 2.90 (2d of AB system, J = 12.68 Hz, 2H, CH 2 N), 3.23 and 3.46 (m, 2H, CH 2 Ph), 3.59 and 3.98 (2d of AB system, J = 13.12 Hz, 2H, PhCH 2 N), 3.58 (m, 1H, CHC=O), 3.52 (m, 1H, CH 3), 5.32 (d, J = 5.0 Hz, 1H, CH-6), 7.29 (m, 10H, Ph). 13 C NMR (CDC 3 ): -4.54 (Si(CH 3

)

2 ), 13.91, 18.28 (SiC(CH 3

)

3 ), 19.37, 20.55, 22.51, 23.03, 23.35, 25.97, 31.26, 31.66, 32.07, 34.50, 36.50, 37.37, 38.52, 41.69, 42.79, 50.09, 54.76, 55.32, 55.40, 58.38, 63.83, 72.56, 85.71, 121.03, 126.75, 127.77, 128.26, 128.29, 130.15, 130.48, 135.47, 137.96, 141.52, 170.52. [00501] Synthesis of Azalactones 5A, 5B, 6A and 6B : General Procedure for the TBS Deprotection 1005021 To a solution of compounds 3A, 3B, 4A or 4B in THF (4 mL) was added a methanolic solution of hydrochloric acid (5%) (3 mL). The mixture was stirred at room temperature during 90 min. A saturated solution of aqueous sodium bicarbonate was added and the aqueous phase extracted four times with ethyl acetate. The organic phase was dried with anhydrous Na 2

SO

4 and evaporated under reduced pressure. The crude compound was purified by flash chromatography using hexane/ethyl acetate/ triethylamine (79:20:1) as eluent for compounds 5A, 5B and hexane/ethyl acetate (3:1) for compounds 6A and 6B respectively.

WO 2012/129673 PCT/CA2012/000316 - 159 1005031 (3S,6R)-3-benzyl-6-[(3p)-3-hydroxyandrost-5-en-17-yll-6 methylmorpholin-2-one (5A) 100504] Yield: 15 mg, 98%; Rf = 0.2 (hexanes/EtOAc, 1:1); IR (film): 3086-3600 (OH and NH), 1720 (C=O of ester). IH NMR (CDC 3 ): 0.87 (s, 3H, CH 3 18), 1.00 (s, 3H, CH 3 -19), 1.31 (s, 3H, CH 3 -21), 1.34-2.60 (unassigned CH and CH 2 ), 2.65 and 2.96 (2d of AB system, J= 13.2 Hz, 2H, CH 2 N), 3.19 (m, 2H, CH 2 -Ph), 3.51 (m, 1H, CH-3), 3.62 (m, IH, CHC=O), 5.34 (d, J = 5.0 Hz, 1H, CH-6), 7.26 (m, 5H, Ph). "C NMR (CDC13): 13.62, 19.38, 20.84, 22.83, 23.69, 23.82, 29.69, 31.26, 31.60, 31.66, 36.45, 37.20, 38.04, 39.87, 42.24, 42.82, 49.97, 51.41, 56.77, 57.65, 58.92, 71.73, 86.88, 121.42, 126.99, 128.75,129.50, 137.34, 140.77, 170.72. [00505] (3R,6R)-3-benzyl-6-[(3 )-3-hydroxyandrost-5-en-17-yll-6 methylmorpholin-2-one (5B) [005061 Yield: 79 mg, 65%; Rf = 0.15 (hexanes/EtOAc, 1:1); IR (film): 3086-3600 (OH and NH), 1713 (C=O of ester). 'H NMR (CDC 3 ): 0.71 (s, 3H, CH 3 18), 0.99 (s, 3H, CH 3 -19), 1.36 (s, 3H, CH 3 -21), 1.39-2.33 (unassigned CH and CH 2 ), 2.72 and 3.06 (2d of AB system, J = 13.8 Hz, 2H, CH 2 N), 3.20 (m, 2H, CH 2 -Ph), 3.55 (m, 1H, CH-3), 3.79 and 3.80 (2d of AB system, J= 4.4 Hz, 1H, CHC=O), 5.36 (d, J = 5.1 Hz, IH, CH-6), 7.30 (m, 5H, Ph). "C NMR (CDC1 3 ): 13.65, 19.34, 20.76, 22.61, 23.27, 23.35, 31.28, 31.56, 31.67, 36.45, 37.26, 37.29, 39.18, 42.06, 42.22, 49.75, 49.95, 55.94, 56.05, 57.42, 71.60, 87.14, 121.39, 127.24, 128.80, 129.82, 137.22, 140.80, 171.13. 100507] (3S,6R)-3,4-dibenzyl-6-[(3p)-3-hydroxyandrost-5-en-17-yll-6 methylmorpholin-2-one (6A) [005081 Yield: 22 mg, 53%; Rf = 0.2 (hexanes/ethyl acetate 3:1); IR (film): 3398 (OH), 1720 (C=0), 1454 (C-0). 'H NMR (CDCl 3 ): 0.78 (s, 3H, CH 3

-

WO 2012/129673 PCT/CA2012/000316 - 160 18), 0.96 (s, 3H, CH 3 -19), 0.97 (s, 3H, CH 3 -21), 0.99-2.17 (unassigned CH and CH 2 ), 2.24 and 2.40 (2d of AB system, J = 12.3 Hz, 2H, CH 2 N), 3.10 and 4.35 (2d of AB system, J= 13.6 Hz, 2H, PhCH 2 N), 3.28 and 3.56 (m, 2H, CH 2 Ph), 3.41 (m, J = 3.0 Hz, IH, CHC=O), 3.49 (m, I H, CH-3), 5.31 (m, 1H, HC-6), 7.36 (m, IOH, Ph). "C NMR (CDCl 3 ): 13.43, 19.35, 20.79, 22.77, 23.53, 23.66, 31.18, 31.59, 31.62, 35.69, 36.41, 37.18, 39.76, 42.23, 42.79, 49.92, 56.39, 56.72, 57.48, 58.22, 66.14, 71.69, 71.72, 84.95, 121.41, 126.62, 127.35, 127.95, 128.08, 128.22, 128.46, 128.51, 130.36, 137.40, 137.51, 140.74, 170.95. 100509] (3R,6R)-3,4-dibenzvl-6-[(3p)-3-hydroxyandrost-5-en-17-yll-6 methylmorpholin-2-one (6B) [00510] Yield: 90 mg, 83%; Rf = 0.15 (hexanes/ethyl acetate 3:1); IR (film): 3402 (OH), 1720 (C=O), 1454 (C-0). 'H NMR (CDC3): 0.57 (s, 3H, CH 3 18), 0.95 (s, 3H, CH 3 -19), 1.24 (s, 3H, CH 3 -21), 1.26-2.19 (unassigned CH and CH 2 ), 2.24 and 2.91 (2d of AB system, J = 12.7 Hz, 2H, CH 2 N), 3.58 and 3.97 (2d of AB system, J = 13.2 Hz, 2H, PhCH 2 N), 3.23 and 3.46 (m, 2H, CH 2 Ph), 3.55 (m, 1H, CHC=O), 3.59 (m, IH, CH-3), 5.36 (d, J = 5.1 Hz, 1H, CH-6), 7.29 (m, 10H, Ph). "C NMR (CDCl 3 ): 13.89, 19.32, 20.56, 22.51, 23.03, 23.38, 31.24, 31.59, 31.62, 34.46, 36.42, 37.23, 38.53, 41.71, 42.26, 50.01, 54.81, 55.23, 55.39, 58.35, 63.80, 71.69, 85.74, 121.53, 126.74, 127.75, 128.26, 128.29, 130.11, 130.45, 135.49, 137.97, 140.78, 170.58. [00511] 170-HSD10 [005121 17p-hydroxysteroid dehydrogenase type 10 (17p-HSD10) is a mitochondrial enzyme involved in estrogen inactivation, androgen activation, p oxidation of fatty acids and isomerisation of bile acids. Since this enzyme uses estradiol (E2) as a substrate, there is evidence that the enzyme contributes to Alzheimer's disease pathogenesis by reducing neuroprotective estrogen levels.

WO 2012/129673 PCT/CA2012/000316 - 161 Moreover, this enzyme plays a significant role in a non-classical androgen synthesis pathway and its expression is up-regulated in certain prostate cancer cells, thus conferring an advantage upon these cells for surviving androgen ablation therapy. Consequently, the inhibition of 17p-HSD1O may provide a new approach to the treatment of these diseases. From the screening study of available molecules in our laboratory, we identified a series of steroid derivatives, showing more than 50% of inhibition for the transformation of estradiol (E2) (1 pM) into estrone (El) by 173 HSD10 when tested at I pM in intact cells. An IC 50 value of 0.55 pM was obtained for RM-532-46, the 3p-androsterone steroid core with the best enzyme inhibition. This inhibitory activity is a good starting point considering that the Km of the enzyme for E2 as a substrate is 43 pM. The obtaining of 17p-HSD1O inhibitors could be useful tools to further elucidate the role of 17p-HSD10 in normal cellular function as well as in regulating steroid hormone levels. [00513] The human 17p-hydroxysteroid dehydrogenase type 10 (17p HSD10) is known for its multiple functions. 17p-HSD10 is a homotetrameric mitochondrial protein and is essentially expressed in the liver and several other tissues including brain and gonads.

6

'

35

'

36 It plays an important role in the metabolism of sex steroid hormones through its 17p-HSD as well as 3a-HSD activities (FIG. 19). This enzyme can inactivate E2, the most potent sex steroid responsible for estrogenic activity in women. A strong link has been established between the development of Alzheimer's disease (AD) and the accumulation of amyloid $-peptide (AP) in the brain.

57

'

5 8 Based on the finding that 17p-HSD1O uses E2 as a substrate, it has been suggested that the enzyme could contribute to AD pathogenesis by reducing neuroprotective E2 levels in the brain. The estrogen-deficient state in AD brain likely promotes neuronal and synaptic loss and impairs hippocampal-dependent learning and memory. He et a1 59 have observed higher levels of 17p-HSD10 in brain regions such as the hippocampus, hypothalamus and amygdale, which are more susceptible to AD. Moreover, E2 exerts neuroprotective effects by its regulation of p-amyloid protein precursor trafficking and metabolism. It was reported that E2 treatment reduces the formation of AP in both in vivo and in vitro experiments.60 Additionally, this enzyme WO 2012/129673 PCT/CA2012/000316 - 162 can catalyze the intracellular oxidation of allopregnanolone, a positive allosteric modulator of GABAA receptors, into 5a-dihydroprogesterone (5a-DHP) in astrocytes. Allopregnanolone rapidly modulates neuronal excitability and has pronounced anxiolytic and anticonvulsant effects in man.

61 He et al 2 reported that the expression of 17p-HSD10 has been greatly up-regulated in the activated astrocytes of AD patients. Taken together, these observations provide evidence that 17p-HSD1O may exacerbate the progress of AD. 100514] 17P-HSDIO also plays a significant role in a non-classical androgen synthesis pathway because it enables prostate cancer cells to generate dihydrotestosterone (DHT) in the absence of testosterone.

63 In fact, this mitochondrial enzyme catalyzes the transformation of 5a-androstane-3a,17p-diol (3a-diol), an almost inactive androgen, into DHT, a potent androgen, by a regioselective oxidation of the 3a-hydroxyl group rather than the 17p-hydroxyl group (FIG. 19). DHT stimulates prostate growth and is implicated in baldness, acne and hirsutism. A higher expression of this dehydrogenase has been demonstrated in certain prostate cancer cells and there is evidence that these high levels of 170-HSDIO might confer an advantage upon these cells for surviving androgen ablation therapy. Thus, inhibition of 17p-HSD10 enzymatic activity provides a new approach to the treatment of AD and prostate cancer in combination with known inhibitors of 5ca-reductase, In addition to potential therapeutic applications, 17P-HSDI0 inhibitors also delineate the role of this steroidogenic enzyme in normal cellular function and in disease pathogenesis. 100515] In one embodiment of the present disclosure, there are included inhibitors of 178-HSDI0, in which the inhibitor has the structure W H X Z .H A N HOi Z Xz WO 2012/129673 PCT/CA2012/000316 - 163 wherein: A is aryl or heteroaryl; W is -C(=O)-, -CH(-OH) or -CH(-COCH 3 ); X is H, alkyl, thioalkyl, halo or alkoxy; Y is -CH 2 -, -C(=O)- or -S(=0) 2 ; Z is H or alkyl. [005161 The 17p-HSDIO inhibitors of the disclosure include compounds such as those shown in Table 6: 100517] Table 6: Inhibition of 17p-HSD10 ([ 14 C]-E2 into [1 4 C]-EI) W H X z NH H N HO Y z Entry Compound x Y Z W Inhibition ()at 1 PiM 1 15a 2-CF 3

SO

2

CH

3 C=O 42 2 -- 2-CF 3

SO

2

CH

3 p-OH 16 3 15b 3-CF 3 so2 CH 3 C=0 30 4 15c 4-CF 3

SO

2

CH

3 C=O 18 5 14b 2-CF 3 S02 H C=O 32 6 14a 3-CF 3 so2 H C=0 3 7 14c 4-CF 3

SO

2 H C=O 14 8 13d 4-CF 3 C=~O CH 3 C=O 22 9 12c 2-CF 3 C=O H C=O 47 10 Ilb 2-CF 3 CH, CH, C=O 25 11 11a 3-CF 3

CH

2

CH

3 C=O 26 12 Ile 4-CF 3

CH

2

CH

3 C=O 24 13 lId 3-CF 3 S CH 2

CH

3 C=O 27 14 1le 3-Cl CH 2

CH

3 C=O 25 15 11f 3-CH 3 O CH 2

CH

3 C=0 21 16 11g H (3-Pyr)* CH 2

CH

3 C=0 21 ----- ------------------------------------------------------------------------------- 17 10b 2-CF 3

CH

2 H C=O 50 WO 2012/129673 PCT/CA2012/000316 - 164 18 loa 3-CF 3

CH

2 H C=O 34 19 10d 3-CF 3 S CH 2 H C=O 29 20 l0e 3-Cl CH 2 H C=O 21 21 lOf 3-CH 3 0 CH 2 H C=O 59 22 log H (3-Pyr)* CH 2 H C=0 37 23 -- H CH 2 H C=O 53

------------------------------

I-------------------------------------------------------

24 7j H CH 2 H C=O 57 25 - H CH 2 H p-COCH 3 11 26 - - - - - 6 27 - - - - - 5 * A 3-pyridyl group instead of a phenyl derivative. ** 4-Androstene instead of 5a-androstane nucleus. 1005181 The IC 5 0 for compound 21 was also determined (the concentration that inhibits 50% of enzymatic activity), and also for the natural substrate of the enzyme E2. As shown in FIG. 20, an IC 5 0 value of 0.55 pM was obtained for compound 21, it was not possible to determine the IC 50 for E2 in the range of concentrations used (0.1-10 pM), suggesting an IC 50 > 10 [M. 100519] FIG. 21 illustrates the inhibition of transfected 17p-HSD10 in intact HEK-293 cells by compound 21 and E2. Compounds were tested at various concentrations to determine IC 50 values. When the error bars are not shown, it is because they are smaller than the symbol. [005201 The chemical synthesis of compounds tested as inhibitors of 17p HSD 10 and shown in Table 6 was reported in previous sections reporting inhibitors of 17p-HSD3. The section corresponding to each tested compound was reported in Table 6. 100521] Enzymatic Assay (Inhibition of 17D-HSD10) 100522] Generation of Stably Transfected Human Embryonic Kidney (HEK)-293 Cells Expressing 170-hydroxysteroid dehydrogenase type 10 WO 2012/129673 PCT/CA2012/000316 - 165 1005231 Cells were cultured in 6-well falcon flasks to approximately 3 x10 5 cells/well in Dubelcco's Modified Eagle's Medium (DMEM) (Life Technologies, Burlington, ON, Canada) supplemented with 10% (vol/vol) of fotal bovine serum (FBS) (HyClone Laboratories, Inc., Logan, UT, USA) at 37 0 C under a 95% air-5% CO 2 humidified atmosphere. Five (5) micrograms of pCMVneo hi7bHSDIO plasmids were transfected using a lipofectin transfection kit (Life Technologies, Burlington, ON, Canada). After 6 h of incubation at 37*C, the transfection medium was removed, and 2 mL DMEM were added. Cells were further cultured for 48 h and then transferred into 10-cm petri dishes and cultured in DMEM containing 700 pg/mL of Geneticin (G418; Wisent, Montreal, QC, Canada) to inhibit the growth of non-transfected cells. Medium containing G418 was changed every 2 days until resistant colonies were observed. 1005241 Cell Culture [00525] Stably transfected HEK-293 cells were cultured in minimum essential medium (MEM) containing non-essential amino acids (0.1 nM), glutamine (2 mM), sodium pyruvate (1 mM), 10% FBS, penicillin (100 IU/mL), streptomycin (100 ig/mL) and G418 (0.7 mg/mL). 1005261 Inhibition of 170-HSD10: In vitro Activity Esing Whole Cells 1005271 HEK-293 cells stably transfected with 17p-HSD10 were seeded at 250 000 cells/well in a 24-well plate at 37 0 C under 95% air 5% CO 2 humidified atmosphere in 990 pL of culture medium. Inhibitor stock solutions were prepared in ethanol and diluted with culture medium. After 24 h, 5 1 iL of these solutions were added to the cells to obtain a final concentration of I ptM for inhibitors. For the most active inhibitors, concentrations of 0.01 pM to 5 pM were tested to determine their

IC

50 value. The final concentration of ethanol in each well was adjusted to 0.5%. Additionally, 5 [tL of a solution containing [ 14 C]17p-estradiol (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) and 17p-estradiol (Sigma- WO 2012/129673 PCT/CA2012/000316 - 166 Aldrich, St. Louis, MO, USA) (1:9) was added to obtain a final concentration of I piM and cells were incubated for 24 h. Each inhibitor was assessed in triplicate. After incubation, the culture medium was removed and labeled steroids (El and E2) were extracted with I mL of diethylether. The organic phases were separated and evaporated to dryness with nitrogen. Residues were dissolved in dichloromethane and dropped on silica gel thin layer chromatography plates (EMD Chemicals Inc., Gibbstown, NJ, USA) and eluted with toluene/acetone (4:1) solvent system. Substrate [14C]-E2 and metabolites [ 14C]-EI were identified by comparing them with reference steroids (E2 and El) and quantified using the Storm 860 system (Molecular Dynamics, Sunnyvale, CA, USA). The percentages of transformation and inhibition were calculated as follow: % transformation = 100 x {14C]-El /([14C]-E2 + [ 14C]-El) and % of inhibition = 100 x (% transformation without inhibitor - % transformation with inhibitor)/% transformation without inhibitor. 1005281 It is to be understood that the specification is not limited in its application to the details of construction and parts as described hereinabove. The specification is capable of other embodiments and of being practiced in various ways. It is also understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Hence, although the present invention has been described hereinabove by way of illustrative embodiments thereof, it can be modified, without departing from the spirit, scope and nature of the subject disclosure as defined in the appended claims.

WO 2012/129673 PCT/CA2012/000316 - 167 REFERENCES (1) Luu-The, V.; Zhang, Y.; Poirier. D.; Labrie, F. Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition. J. Steroid Biochem. Mol. Biol. 1995, 55, 581-587. (2) Simard, J.; Vincent, A.; Duchesne, R.; Labrie, F. Full estrogenic activity of C19 delta 5 adrenal steroids in rat pituitary lactotrophs and somatotrophs. Mol. Cell. Endocrinol. 1988, 55, 233-242. (3) Theobald, A. J. Management of advanced breast cancer with endocrine therapy: the role of the primary healthcare team. Int. J. Clin. Pract. 2000, 54, 665-669. (4) Dizerega, G. S.; Barber, D. L.; Hodgen, G. D. Endometriosis: role of ovarian steroids in initiation, maintenance, and suppression. Fertil. Steril. 1980, 33, 649 653. (5) Penning, T. M. 17-hydroxysteroid dehydrogenase: inhibitors and inhibitor design, Endocr. Relat. Cancer, 1996, 3, 41-56. (6) Poirier, D. Advances in Development of Inhibitors of 17J-Hydroxysteroid Dehydrogenases. Anticancer Agents Med Chem., 2009, 9, 642-660. (7) Moeller, G.; Adamski, J. Integrated view on 17p-hydroxysteroid dehydrogenase. Mol. Cell. Endocrinol. 2009, 301, 7-19. (8) Poirier, D. Contribution to the development of inhibitors of 17p-hydroxysteroid dehydrogenase type I and 7: Key tools for studying and treating estrogen dependent diseases. J. Steroid Biochem. Mol. Biol., 2011, 125, 83-94. (9) Poirier, D. 17p-Hydroxysteroid dehydrogenase inhibitors: a patent review. Expert. Opin. Ther. Patents, 2010, 20, 1123-1145. (10) Poirier, D. Inhibitors of 17p-hydroxysteroid dehydrogenase. Curr. Med. Chem, 2003, 10, 453-477. (11) Tremblay, M. R.; Poirier, D. Overview of a rational approach to design type 117 beta-hydroxysteroid dehydrogenase inhibitors without estrogenic activity: chemical synthesis and biological evaluation. J. Steroid Biochem. Mol. Biol, 1998, 66, 179-191. (12) Laplante, Y.; Cadot, C.; Fournier, M. C.; Poirier, D. Estradiol and Estrone C-16 derivatives as inhibitors of type I1 17-hydroxysteroid dehydrogenase: Blocking of ER+ breast cancer cell proliferation induced by estrone. Bioorg. Med. Chem. 2008, 16, 1849-1860. (13) Mazumdar, M.; Fournier, D.; Zhu, D. W.; Cadot, C.; Poirier, D.; Lin, S. X. Binary and ternary crystal structure analyses of a novel inhibitor with 17p-HSD type 1: a lead compound for breast cancer therapy. Biochem. J. 2009, 10, 357 366. (14) http://www.cancer.org/Cancer-ProstateCancer/DetailedGuide/prostate-cancer key-statistics.

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Claims

1. An inhibitor of 17B-HSDI.

2. The inhibitor of claim 1, wherein the inhibitor has the structure: OH R NH2 wherein R is -OH, halo, -NH 2 , alkyl, alkoxy, aralkyl, carboxy, -CH 2 -heterocyclyl, heterocyclyloyl, -C(O)N(R')(R") or -B(OH) 2 , wherein R' and R" are independently or simultanesouly H or alkyl, or R' and R" are joined together, along with the nitrogen atom to which they are attached, to form a heterocyclic ring, or a pharaceutically acceptable salt or tautomer thereof.

3. The inhibitor of claim 2, wherein the inhibitor has the structure OH HO EH NH2 Br or a pharmaceutically acceptable salt thereof.

4. The inhibitor of claim 3, wherein the inhibitor has the structure WO 2012/129673 PCT/CA2012/000316 - 173 OH H H NH2 Br * HCI

5. The inhibitor of claim 1, wherein the inhibitor has the structure: OH HO Br or a pharmaceutically acceptable salt thereof.

6. An inhibitor of 17B-HSD3.

7. The inhibitor of claim 6, wherein the inhibitor has the structure: 0 H R OH H wherein R is heterocyclyl or -NRaRb, wherein Ra and Rb are each independently alkyl or aralkyl, or a pharmaceutically acceptable salt or tautomer thereof.

8. The inhibitor of claim 6, wherein the inhibitor has the structure: WO 2012/129673 PCT/CA2012/000316 -174 H OHHf wherein: -- N N -N N Xis or Y is -CH 2 -, -C(O)- or -S(O) 2 -; and Z is cycloalkyl, aryl or heterocyclyl, or a pharmaceutically acceptable salt or tautomer thereof.

9. The inhibitor of claim 8, wherein the inhibitor has the structure 0 H S -N N CF 3 or a pharmaceutically acceptable salt thereof

10. The inhibitor of claim 9, wherein the inhibitor has the structure 0 H r -- SN N. N HO H CF 3 HCI WO 2012/129673 PCT/CA2012/000316 -175

11. The inhibitor of claim 6, wherein the inhibitor has the structure: 0 H R2 R 2 1 0 wherein: R' is alkyl, aralkyl, heterocyclyl, cycloalkyl, -CH 2 CH 2 S(O) 2 -aryl, heterocyclyl, -CH2CH 2 CH(O-acyl)CH 2 -heterocyclyl, or -CH 2 CH 2 NRaRb, wherein Ra and Rb are each independently alkyl, cycloalkyl, acyl, or aralkyl; and each R 2 is independently hydrogen or alkyl, or a pharmaceutically acceptable salt or tautomer thereof.

12. The inhibitor of claim 11, wherein the inhibitor has the structure 0 0 R 2 H wherein, Xi is alkyl, R2 R2 is independently hydrogen or alkyl, R 3 is hydrogen, alkyl or aralkyl, or a pharmaceutically acceptable salt or tautomer thereof.

13. The inhibitor of claim 6, wherein the inhibitor has the structure: WO 2012/129673 PCT/CA2012/000316 - 176 a0 R2 H R 4 R 2 N R5 wherein, R2 is independently hydrogen or alkyl, R 4 is hydrogen or aralkyl, R 5 is hydrogen, alkyl or aralkyl, or a pharmaceutically acceptable salt or tautomer thereof.

14. An inhibitor of 17B-HSDI and 17B-HSD3.

15. The inhibitor of claim 14, wherein the inhibitor has the structure Xa or Xb: 0 00 N R6 l ,r \ N H Ry7 H R7 HH H Xa Xb wherein: R 6 and R 7 are independently or simultaneously H, alkyl, cycloalkyl, aryl, aralkyl, or heterocyclyl, or a pharmaceutically acceptable salt or tautomer thereof.

16. An inhibitor of 17B-HSD10.

17. The inhibitor of claim 16, wherein the inhibitor has the structure: WO 2012/129673 PCT/CA2012/000316 - 177 W Z H N H Y N HO z wherein: A is aryl or heteroaryl; W is -C(=O)-, -CH(-OH) or -CH(-COCH 3 ); X is H, alkyl, thioalkyl, halo or alkoxy; Y is -CH 2 -, -C(=O)- or -S(=0)2; Z is H or alkyl, or a pharmaceutically acceptable salt or tautomer thereof.

18. A method of treating cancer in a subject comprising administering to said subject a compound as shown in claims 1 to 17.

19. The method of claim 18, wherein said cancer is an estrogen-sensitive cancer.

20. The method of claim 19, wherein said cancer is breast cancer.

21. The method of claim 20, wherein said compound is a compound of claims 1 to 5 or 14 to 15.

22. The method of claim 18, wherein said cancer is an androgen-sensitive cancer.

23. The method of claim 18, wherein said cancer is prostate cancer.

24. The method of claim 23, wherein said compound is a compound of any one of claims 1 to 17.

25. The method of claim 18, further comprising treating said subject with a secondary cancer therapy.

26. The method of claim 25, wherein said secondary cancer therapy is WO 2012/129673 PCT/CA2012/000316 - 178 selected from the group consisting of chemotherapy, toxin therapy, radiation therapy, hormone or anti-hormone therapy, surgery, cryotherapy, or immunotherapy.

27. A method of treating a non-cancerous disease is a subject comprising administering to said subject a compound as shown in claims 1 to 17.

28. The method of claim 27, wherein said non-cancerous disease is endometriosis.

29. The method of claim 28, wherein said compound is a compound of claims 1 to 5 or 14 to 15.

30. The method of claim 27, wherein said non-cancerous disease is benign prostatic hyperplasia.

31. The method of claim 30, wherein said compound is a compound of claims 6 to 15.

32. The method of claim 27, wherein said non-cancerous disease is Alzheimer's disease.

33. The method of claim 32, wherein said compound is a compound of claims 9 or 10.

34. The method of claim 27, further comprising treating said subject with a secondary therapy.

35. The method of any of claims 18 to 34, further comprising administering said compound at least a second time.

36. The method of any of claims 18 to 34, wherein said compound is administered intravenously, intra-arterially, subcutaneously, topically, or intramuscularly.

37. The method of any of claims 18 to 34, wherein said compound is administered systemically, regionally to a tumor/disease site, locally to a WO 2012/129673 PCT/CA2012/000316 - 179 tumor/disease site, into tumor/tissue vasculature or intratumorally.

38. The method of any of claims 18 to 26, wherein said cancer is multi drug resistant.

39. The method of any of claims 18 to 26, wherein said cancer is metastatic.

40. The method of any of claims 18 to 26, wherein said cancer is recurrent.

41. The method of any of claims 18 to 26, wherein treating comprises inhibiting cancer growth, killing cancer cells, reducing tumor burden, reducing tumor size, improving said subject's quality of life or prolonging said subject's length of life.

42. The method of any of claims 18 to 26, wherein the cancer is lung cancer, brain cancer, head & neck cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovaran cancer, testicular cancer, skin cancer or esophageal cancer.

43. The method of any of claims 18 to 34, wherein said subject is a human.

44. The method of any of claims 18 to 34, wherein said subject is a non human animal.

45. A radiolabelled compound comprising the structure OH x 0 NH, wherein X is I 1, I 1 or Br 76 , or a pharmaceutically acceptable salt or tautomer thereof

46. A radiolabelled compound comprising the structure WO 2012/129673 PCT/CA2012/000316 -180 OH x 123 125 131 76 wherein X is I , I , I or Br , or a pharmaceutically acceptable salt or tautomer thereof.

47. The radiolabelled compound of claim 45, for radioimaging or treating breast cancer or prostate cancer.